Color Atlas of Hematology - Practical Microscopic and Clinical ...

iiiColor Atlas of HematologyPractical Microscopic and Clinical DiagnosisHarald Theml, M.D.Professor, Private PracticeHematology/OncologyMunich, GermanyHeinz Diem, M.D.Klinikum GrosshadernInstitute of Clinical ChemistryMunich, GermanyTorsten Haferlach, M.D.Professor, Klinikum GrosshadernLaboratory for Leukemia DiagnosticsMunich, Germany2nd revised edition262 color illustrations32 tablesThiemeStuttgart · New York

ivLibrary of Congress Cataloging-in-PublicationData is available from the publisherThis book is an authorized revisedtranslation of the 5th German editionpublished and copyrighted 2002 byThieme Verlag, Stuttgart, Germany.Title of the German edition:Taschenatlas der HämatologieTranslator: Ursula Peter-Czichi PhD,Atlanta, GA, USA1st German edition 19832nd German edition 19863rd German edition 19914th German edition 19985th German edition 20021st English edition 19851st French edition 19852nd French edition 20001st Indonesion edition 19891st Italian edition 19841st Japanese edition 1997Important note: Medicine is an everchangingscience undergoing continualdevelopment. Research and clinical experienceare continually expanding ourknowledge, in particular our knowledge ofproper treatment and drug therapy. Insofaras this book mentions any dosage or application,readers may rest assured that theauthors, editors, and publishers have madeevery effort to ensure that such referencesare in accordance with the state of knowledgeat the time of production of thebook.Nevertheless, this does not involve, imply,or express any guarantee or responsibilityon the part of the publishers in respect toany dosage instructions and forms of applicationsstated in the book. Every user is requestedto examine carefully the manufacturers’leaflets accompanying each drugand to check, if necessary in consultationwith a physician or specialist, whether thedosage schedules mentioned therein or thecontraindications stated by the manufacturersdiffer from the statements made inthe present book. Such examination is particularlyimportant with drugs that areeither rarely used or have been newly releasedon the market. Every dosageschedule or every form of application usedis entirely at the user’s own risk and responsibility.The authors and publishers requestevery user to report to the publishersany discrepancies or inaccuracies noticed. 2004 Georg Thieme VerlagRüdigerstraße 14, 70469 Stuttgart,Germanyhttp://www.thieme.deThieme New York, 333 Seventh Avenue,New York, NY 10001 USAhttp://www.thieme.comCover design: Cyclus, StuttgartTypesetting and printing in Germany byDruckhaus Götz GmbH, LudwigsburgISBN 3-13-673102-6 (GTV)ISBN 1-58890-193-9 (TNY) 1 2 3 4 5Some of the product names, patents, andregistered designs referred to in this bookare in fact registered trademarks or proprietarynames even though specific referenceto this fact is not always made in thetext. Therefore, the appearance of a namewithout designation as proprietary is not tobe construed as a representation by thepublisher that it is in the public domain.This book, including all parts thereof, is legallyprotected by copyright. Any use, exploitation,or commercialization outsidethe narrow limits set by copyright legislation,without the publisher’s consent, is illegaland liable to prosecution. This applies inparticular to photostat reproduction, copying,mimeographing, preparation of microfilms,and electronic data processing andstorage.

vPrefaceOur Current EditionAlthough this is the second English edition of our hematology atlas, thisedition is completely new. As an immediate sign of this change, there arenow three authors. The completely updated visual presentation uses digitalimages, and the content is organized according to the most up-to-datemorphological classification criteria.In this new edition, our newly formed team of authors from Munich(the “Munich Group”) has successfully shared their knowledge with you.Heinz Diem and Torsten Haferlach are nationally recognized as lecturersof the diagnostics curriculum of the German Association for Hematologyand Oncology.GoalsMost physicians are fundamentally “visually oriented.” Apart from immediatepatient care, the microscopic analysis of blood plays to this preference.This explains the delight and level of involvement on the part ofpractitioners in the pursuit of morphological analyses.Specialization notwithstanding, the hematologist wants to preservethe opportunity to perform groundbreaking diagnostics in hematology forthe general practitioner, surgeon, pediatrician, the MTA technician, and allmedical support personnel. New colleagues must also be won to thecause. Utmost attention to the analysis of hematological changes is essentialfor a timely diagnosis.Even before bone marrow cytology, cytochemistry, or immunocytochemistry,information based on the analysis of blood is of immediate relevancein the doctor’s office. It is central to the diagnosis of the diseases ofthe blood cell systems themselves, which make their presence knownthrough changes in blood components.The exhaustive quantitative and qualitative use of hematological diagnosticsis crucial. Discussions with colleagues from all specialties andteaching experience with advanced medical students confirm its importance.In cases where a diagnosis remains elusive, the awareness of thenext diagnostic step becomes relevant. Then, further investigationthrough bone marrow, lymph node, or organ tissue cytology can yield firmresults. This pocket atlas offers the basic knowledge for the use of thesetechniques as well.

viPrefaceOrganizationReflecting our goals, the inductive organization proceeds from simple tospecialized diagnostics. By design, we subordinated the description of thebone marrow cytology to the diagnostic blood analysis (CBC). However,we have responded to feedback from readers of the previous editions andhave included the principles of bone marrow diagnostics and nonambiguousclinical bone marrow findings so that frequent and relevantdiagnoses can be quickly made, understood, or replicated.The nosology and differential diagnosis of hematological diseases arepresented to you in a tabular form. We wanted to offer you a pocketbookfor everyday work, not a reference book. Therefore, morphological curiosities,or anomalies, are absent in favor of a practical approach to morphology.The cellular components of organ biopsies and exudates arebriefly discussed, mostly as a reminder of the importance of these tests.The images are consistently photographed as they normally appear inmicroscopy (magnification 100 or 63 with oil immersion lens, occasionallymaster-detail magnification objective 10 or 20). Even thoughsurprising perspectives sometimes result from viewing cells at a highermagnification, the downside is that this by no means facilitates the recognitionof cells using your own microscope.Instructions for the Use of this AtlasThe organization of this atlas supports a systematic approach to the studyof hematology (see Table of Contents). The index offers ways to answerdetailed questions and access the hematological terminology with referencesto the main description and further citations.The best way to become familiar with your pocket atlas is to first have acursory look through its entire content. The images are accompanied byshort legends. On the pages opposite the images you will find correspondingshort descriptive texts and tables. This text portion describes cell phenomenaand discusses in more detail further diagnostic steps as well asthe diagnostic approach to disease manifestations.AcknowledgmentsTwenty years ago, Professor Herbert Begemann dedicated the foreword tothe first edition of this hematology atlas. He acknowledged that—beyondcell morphology—this atlas aims at the clinical picture of patients. We aregrateful for being able to continue this tradition, and for the impulses fromour teachers and companions that make this possible.We thank our colleagues: J. Rastetter, W. Kaboth, K. Lennert, H. Löffler,H. Heimpel, P.M. Reisert, H. Brücher, W. Enne, T. Binder, H.D. Schick, W.Hiddemann, D. Seidel.Munich, January 2004Harald Theml, Heinz Diem, Torsten Haferlach

viiContentsPhysiology and Pathophysiology of Blood Cells:Methods and Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Introduction to the Physiology and Pathophysiology of theHematopoietic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Cell Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Principles of Regulation and Dysregulation in the Blood CellSeries and their Diagnostic Implications . . . . . . . . . . . . . . . . . . . . . . . . 7Procedures, Assays, and Normal Values . . . . . . . . . . . . . . . . . . . . . . . 9Taking Blood Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Erythrocyte Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Hemoglobin and Hematocrit Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Calculation of Erythrocyte Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 10Red Cell Distribution Width (RDW) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Reticulocyte Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Leukocyte Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Thrombocyte Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Quantitative Normal Values and Distribution of Cellular BloodComponents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15The Blood Smear and Its Interpretation (Differential Blood Count,DBC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Significance of the Automated Blood Count . . . . . . . . . . . . . . . . . . . . . 19Bone Marrow Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Lymph Node Biopsy and Tumor Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . 23Step-by-Step Diagnostic Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Normal Cells of the Blood and Hematopoietic Organs . 29The Individual Cells of Hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . 30Immature Red Cell Precursors: Proerythroblasts and BasophilicErythroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Mature Red Blood Precursor Cells: Polychromatic and OrthochromaticErythroblasts (Normoblasts) and Reticulocytes . . . . . . . 32Immature White Cell Precursors: Myeloblasts and Promyelocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

viiiContentsPartly Mature White Cell Precursors: Myelocytes and Metamyelocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Mature Neutrophils: Band Cells and Segmented Neutrophils . . . . . 38Cell Degradation, Special Granulations, and Nuclear Appendagesin Neutrophilic Granulocytes and Nuclear Anomalies . . . . . . . . . . . . 40Eosinophilic Granulocytes (Eosinophils) . . . . . . . . . . . . . . . . . . . . . . . . 44Basophilic Granulocytes (Basophils) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Monocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Lymphocytes (and Plasma Cells) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Megakaryocytes and Thrombocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Bone Marrow: Cell Composition and Principles of Analysis . . . . 52Bone Marrow: Medullary Stroma Cells . . . . . . . . . . . . . . . . . . . . . . . . . 58Abnormalities of the White Cell Series . . . . . . . . . . . . . . . . . . 61Predominance of Mononuclear Round to Oval Cells . . . . . . . . . . . 63Reactive Lymphocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66Examples of Extreme Lymphocytic Stimulation: InfectiousMononucleosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Diseases of the Lymphatic System (Non-Hodgkin Lymphomas) . . . 70Differentiation of the Lymphatic Cells and Cell Surface MarkerExpression in Non-Hodgkin Lymphoma Cells . . . . . . . . . . . . . . . . . 72Chronic Lymphocytic Leukemia (CLL) and Related Diseases . . . . 74Lymphoplasmacytic Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Facultative Leukemic Lymphomas (e.g., Mantle Cell Lymphomaand Follicular Lymphoma) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Lymphoma, Usually with Splenomegaly (e.g., Hairy Cell Leukemiaand Splenic Lymphoma with Villous Lymphocytes) . . . . . . . 80Monoclonal Gammopathy (Hypergammaglobulinemia), MultipleMyeloma*, Plasma Cell Myeloma, Plasmacytoma . . . . . . . . . 82Variability of Plasmacytoma Morphology . . . . . . . . . . . . . . . . . . . . . 84Relative Lymphocytosis Associated with Granulocytopenia(Neutropenia) and Agranulocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86Classification of Neutropenias and Agranulocytoses . . . . . . . . . . . 86Monocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88Acute Leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Morphological and Cytochemical Cell Identification . . . . . . . . . . . 91Acute Myeloid Leukemias (AML) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95Acute Erythroleukemia (FAB Classification Type M 6 ) . . . . . . . . . . 100Acute Megakaryoblastic Leukemia(FAB Classification Type M 7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102AML with Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102Hypoplastic AML . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

ContentsixAcute Lymphoblastic Leukemia (ALL) . . . . . . . . . . . . . . . . . . . . . . . . 104Myelodysplasia (MDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106Prevalence of Polynuclear (Segmented) Cells . . . . . . . . . . . . . . . . . 110Neutrophilia without Left Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110Reactive Left Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112Chronic Myeloid Leukemia and Myeloproliferative Syndrome(Chronic Myeloproliferative Disorders, CMPD) . . . . . . . . . . . . . . . . . . 114Steps in the Diagnosis of Chronic Myeloid Leukemia . . . . . . . . . . 116Blast Crisis in Chronic Myeloid Leukemia . . . . . . . . . . . . . . . . . . . . . 120Osteomyelosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122Elevated Eosinophil and Basophil Counts . . . . . . . . . . . . . . . . . . . . . . . 124Erythrocyte and Thrombocyte Abnormalities . . . . . . . . . . . 127Clinically Relevant Classification Principle for Anemias: MeanErythrocyte Hemoglobin Content (MCH) . . . . . . . . . . . . . . . . . . . . . . . 128Hypochromic Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128Iron Deficiency Anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128Hypochromic Infectious or Toxic Anemia (Secondary Anemia) . . . 134Bone Marrow Cytology in the Diagnosis of Hypochromic Anemias. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136Hypochromic Sideroachrestic Anemias (Sometimes Normochromicor Hyperchromic) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137Hypochromic Anemia with Hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . 138Thalassemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138Normochromic Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140Normochromic Hemolytic Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140Hemolytic Anemias with Erythrocyte Anomalies . . . . . . . . . . . . . . . . 144Normochromic Renal Anemia (Sometimes Hypochromic orHyperchromic) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Bone Marrow Aplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Pure Red Cell Aplasia (PRCA, Erythroblastopenia) . . . . . . . . . . . . . 146Aplasias of All Bone Marrow Series (Panmyelopathy, Panmyelophthisis,Aplastic Anemia) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148Bone Marrow Carcinosis and Other Space-Occupying Processes . . 150Hyperchromic Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Erythrocyte Inclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156Hematological Diagnosis of Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

xContentsPolycythemia Vera (Erythremic Polycythemia)and Erythrocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162Thrombocyte Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164Thrombocytopenias Due to Increased Demand(High Turnover) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164Thrombocytopenias Due to Reduced Cell Production . . . . . . . . . . 168Thrombocytosis (Including Essential Thrombocythemia) . . . . . . . . 170Essential Thrombocythemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170Cytology of Organ Biopsies and Exudates . . . . . . . . . . . . . . . 173Lymph Node Cytology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174Reactive Lymph Node Hyperplasia and Lymphogranulomatosis(Hodgkin Disease) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176Sarcoidosis and Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180Non-Hodgkin Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182Metastases of Solid Tumors in Lymph Nodes or SubcutaneousTissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182Branchial Cysts and Bronchoalveolar Lavage . . . . . . . . . . . . . . . . . . 184Branchial Cysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184Cytology of the Respiratory System, Especially BronchoalveolarLavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184Cytology of Pleural Effusions and Ascites . . . . . . . . . . . . . . . . . . . . . 186Cytology of Cerebrospinal Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

2Physiology and Pathophysiology of Blood CellsIntroduction to the Physiology andPathophysiology of the HematopoieticSystemThe reason why quantitativeand qualitative diagnosisbased on the cellularcomponents of the blood isso important is that bloodcells are easily accessibleindicators of disturbancesin their organs of origin ordegradation—which aremuch less easily accessible.Thus, disturbances in theerythrocyte, granulocyte,and thrombocyte seriesallow important conclusionsto be drawn aboutbone marrow function, justas disturbances of the lymphaticcells indicate reactionsor disease states ofthe specialized lymphopoieticorgans (basically,the lymph nodes, spleen,and the diffuse lymphaticintestinal organ).NK cellsPluripotentlymphaticstem cellsT-lymphopoiesisT-lymphoblastsPluripotent hemato-B-lymphopoiesisB-lymphoblastsCell SystemsAll blood cells derive from acommon stem cell. Underthe influences of local andhumoral factors, stem cellsdifferentiate into differentNK cellsT-lymphocytesB-lymphocytesFig. 1 Model of cell lineages in hematopoiesisPlasma cells

Introduction to the Physiology and Pathophysiology3cell lines (Fig. 1). Erythropoiesis and thrombopoiesis proceed independentlyonce the stem cell stage has been passed, whereas monocytopoiesisand granulocytopoiesis are quite closely “related.” Lymphocytopoiesisis the most independent among the remaining cell series. Granulocytes,monocytes, and lymphocytes are collectively called leukocytes (whiteblood cells), a term that has been retained since the days before stainingOmnipotentstem cellsPluripotent myeloidstem cellspoietic stem cellsBasophilsEosinophilsGranulopoiesismonopoiesisThrombopoiesisErythropoiesisMonopoiesisGranulopoiesisMonoblastsMyeloblastsProerythroblastsMegakaryoblastsBasophilic Eosinophilicpromyelocytes promyelocytesPromonocytesPromyelocytesMyelocytesMetamyelocytesCells with band nucleiMegakaryocytesErythroblastsBasophilicsegmentedgranulocytesEosinophilicsegmentedgranulocytesMonocytesNeutrophilicgranulocyteswith segmentednucleiThrombocytesErythrocytesMacrophages

4Physiology and Pathophysiology of Blood Cellsmethods were available, when the only distinction that could be madewas between erythrocytes (red blood cells) and the rest.All these cells are eukaryotic, that is, they are made up of a nucleus,sometimes with visible nucleoli, surrounded by cytoplasm, which may includevarious kinds of organelles, granulations, and vacuoles.Despite the common origin of all the cells, ordinary light microscopyreveals fundamental and characteristic differences in the nuclear chromatinstructure in the different cell series and their various stages ofmaturation (Fig. 2).The developing cells in the granulocyte series (myeloblasts and promyelocytes),for example, show a delicate, fine “net-like” (reticular) structure.Careful microscopic examination (using fine focus adjustment toview different depth levels) reveals a detailed nuclear structure that resemblesfine or coarse gravel (Fig. 2 a). With progressive stages of nuclearmaturation in this series (myelocytes, metamyelocytes, and band or staffcells), the chromatin condenses into bands or streaks, giving the nucleus—which at the same time is adopting a characteristic curved shape—aspotted and striped pattern (Fig. 2 b).Lymphocytes, on the other hand—particularly in their circulatingforms—always have large, solid-looking nuclei. Like cross-sectionsthrough geological slate, homogeneous, dense chromatin bands alternatewith lighter interruptions and fissures (Fig. 2 c).Each of these cell series contains precursors that can divide (blast precursors)and mature or almost mature forms that can no longer divide; themorphological differences between these correspond not to steps in mito-abVacuolesLobed nucleus withbanded chromatinstructureNucleus (withdelicate reticularchromatin structure)CytoplasmNucleolusCytoplasmic granulescCoarse chromatinstructureFig. 2 Principles ofcell structure with examplesof differentnuclear chromatinstructure. a Cell of themyeloblast to promyelocytetype. b Cellof the myelocyte tostaff or band cell type.c Cell of the lymphocytetype withcoarsely structuredchromatin

Introduction to the Physiology and Pathophysiology5sis, but result from continuous “maturation processes” of the cell nucleusand cytoplasm. Once this is understood, it becomes easier not to be toorigid about morphological distinctions between certain cell stages. Theblastic precursors usually reside in the hematopoietic organs (bone marrowand lymph nodes). Since, however, a strict blood–bone marrow barrierdoes not exist (blasts are kept out of the bloodstream essentially onlyby their limited plasticity, i.e., their inability to cross the diffusion barrierinto the bloodstream), it is in principle possible for any cell type to befound in peripheral blood, and when cell production is increased, thestatistical frequency with which they cross into the bloodstream will naturallyrise as well. Conventionally, cells are sorted left to right from immatureto mature, so an increased level of immature cells in the bloodstreamcauses a “left shift” in the composition of a cell series—although itmust be said that only in the precursor stages of granulopoiesis are the cellmorphologies sufficiently distinct for this left shift to show up clearly.The distribution of white blood cells outside their places of origin cannotbe inferred simply from a drop of capillary blood. This is because themajority of white cells remain out of circulation, “marginated” in theepithelial lining of vessel walls or in extravascular spaces, from wherethey may be quickly recruited back to the bloodstream. This phenomenonexplains why white cell counts can vary rapidly without or before anychange has taken place in the rate of their production.Cell functions. A brief indication of the functions of the various cell groupsfollows (see Table 1).Neutrophil granulocytes with segmented nuclei serve mostly todefend against bacteria. Predominantly outside the vascular system, in “inflamed”tissue, they phagocytose and lyse bacteria. The blood merelytransports the granulocytes to their site of action.The function of eosinophilic granulocytes is defense against parasites;they have a direct cytotoxic action on parasites and their eggs and larvae.They also play a role in the down-regulation of anaphylactic shock reactionsand autoimmune responses, thus controlling the influence of basophiliccells.The main function of basophilic granulocytes and their tissue-boundequivalents (tissue mast cells) is to regulate circulation through the releaseof substances such as histamine, serotonin, and heparin. These tissuehormones increase vascular permeability at the site of various local antigenactivity and thus regulate the influx of the other inflammatory cells.The main function of monocytes is the defense against bacteria, fungi,viruses, and foreign bodies. Defensive activities take place mostly outsidethe vessels by phagocytosis. Monocytes also break down endogenous cells(e.g., erythrocytes) at the end of their life cycles, and they are assumed toperform a similar function in defense against tumors. Outside the bloodstream,monocytes develop into histiocytes; macrophages in the

6Physiology and Pathophysiology of Blood Cellsendothelium of the body cavities; epithelioid cells; foreign body macrophages(including Langhans’ giant cells); and many other cells.Lymphocytes are divided into two major basic groups according tofunction.Thymus-dependent T-lymphocytes, which make up about 70% oflymphocytes, provide local defense against antigens from organic and inorganicforeign bodies in the form of delayed-type hypersensitivity, as classicallyexemplified by the tuberculin reaction. T-lymphocytes are dividedinto helper cells and suppressor cells. The small group of NK (naturalkiller) cells, which have a direct cytotoxic function, is closely related to theT-cell group.The other group is the bone-marrow-dependent B-lymphocytes or B-cells, which make up about 20% of lymphocytes. Through their developmentinto immunoglobulin-secreting plasma cells, B-lymphocytes are responsiblefor the entire humoral side of defense against viruses, bacteria,and allergens.Table 1Cells in a normal peripheral blood smear and their physiological rolesCell type Function Count(% of leukocytes)Neutrophilic bandgranulocytes (bandneutrophil)Neutrophilic segmentedgranulocyte (segmentedneutrophil)Lymphocytes(B- and T-lymphocytes,morphologically indistinguishable)MonocytesEosinophilic granulocytesBasophilic granulocytesPrecursors of segmented cellsthat provide antibacterialimmune responsePhagocytosis of bacteria;migrate into tissue for this purposeB-lymphocytes (20% of ⎫lymphocytes) mature and ⎪form plasma cells antibody⎪⎪production.⎬T-lymphocytes (70%): cyto-⎪toxic defense against viruses, ⎪foreign antigens, and tumors. ⎭Phagocytosis of bacteria, protozoa,fungi, foreign bodies.Transformation in target tissueImmune defense against parasites,immune regulationRegulation of the response tolocal inflammatory processes0–4%50–70%20–50%2–8%1–4%0–1%

Introduction to the Physiology and Pathophysiology7Erythrocytes are the oxygen carriers for all oxygen-dependent metabolicreactions in the organism. They are the only blood cells without nuclei,since this allows them to bind and exchange the greatest number ofO 2 molecules. Their physiological biconcave disk shape with a thick rimprovides optimal plasticity.Thrombocytes form the aggregates that, along with humoral coagulationfactors, close up vascular lesions. During the aggregation process, inaddition to the mechanical function, thrombocytic granules also releasefactors that promote coagulation.Thrombocytes develop from polyploid megakaryocytes in the bonemarrow. They are the enucleated, fragmented cytoplasmic portions ofthese progenitor cells.Principles of Regulation and Dysregulation in theBlood Cell Series and their Diagnostic ImplicationsQuantitative and qualitative equilibrium between all blood cells is maintainedunder normal conditions through regulation by humoral factors,which ensure a balance between cell production (mostly in the bone marrow)and cell degradation (mostly in the spleen, liver, bone marrow, andthe diffuse reticular tissue).Compensatory increases in cell production are induced by cell loss or increasedcell demand. This compensatory process can lead to qualitativechanges in the composition of the blood, e.g., the occurrence of nucleatedred cell precursors compensating for blood loss or increased oxygen requirement,or following deficiency of certain metabolites (in the restitutionphase, e.g., during iron or vitamin supplementation). Similarly,during acute immune reactions, which lead to an increased demand forcells, immature leukocyte forms may appear (“left shift”).Increased cell counts in one series can lead to suppression of cell productionin another series. The classic example is the suppression of erythrocyteproduction (the pathomechanical details of which are incompletelyunderstood) during infectious/toxic reactions, which affect the white cells(“infectious anemia”).Metabolite deficiency as a pathogenic stimulus affects the erythrocyte seriesfirst and most frequently. Although other cell series are also affected,this series, with its high turnover, is the one most vulnerable to metabolitedeficiencies. Iron deficiency, for example, rapidly leads to reducedhemoglobin in the erythrocytes, while vitamin B 12 and/or folic acid deficiencywill result in complex disturbances in cell formation. Eventually,these disturbances will start to show effects in the other cell series as well.

8Physiology and Pathophysiology of Blood CellsToxic influences on cell production usually affect all cell series. The effectsof toxic chemicals (including alcohol), irradiation, chronic infections, ortumor load, for example, usually lead to a greater or lesser degree of suppressionin all the blood cell series, lymphocytes and thrombocytes beingthe most resistant. The most extreme result of toxic effects is panmyelophthisis(the synonym “aplastic anemia” ignores the fact that the leukocyteand thrombocyte series are usually also affected).Autoimmune and allergic processes may selectively affect a single cell series.Results of this include “allergic” agranulocytosis, immunohemolyticanemia, and thrombocytopenia triggered by either infection or medication.Autoimmune suppression of the pluripotent stem cells can alsooccur, causing panmyelophthisis.Malignant dedifferentiation can basically occur in cells of any lineage atany stage where the cells are able to divide, causing chronic or acute clinicalmanifestations. These deviations from normal differentiation occur mostfrequently in the white cell series, causing “leukemias.” Recent data indicatethat in fact in these cases the remaining cell series also become distorted,perhaps via generalized atypical stem cell formation. Erythroblastosis,polycythemia, and essential thrombocythemia are examples showingthat malignant processes can also manifest themselves primarily inthe erythrocyte or thrombocyte series.Malignant “transformations” always affect blood cell precursors thatare still capable of dividing, and the result is an accumulation of identical,constantly self-reproducing blastocytes. These are not necessarily alwaysobserved in the bloodstream, but can remain in the bone marrow. That iswhy, in “leukemia,” it is often not the number of cells, but the increasinglack of normal cells that is the indicative hematological finding.All disturbances of bone marrow function are accompanied by quantitativeand/or qualitative changes in the composition of blood cells orblood proteins. Consequently, in most disorders, careful analysis ofchanges in the blood together with clinical findings and other laboratorydata produces the same information as bone marrow cytology. The relationshipbetween the production site (bone marrow) and the destination(the blood) is rarely so fundamentally disturbed that hematological analysisand humoral parameters will not suffice for a diagnosis. This is virtuallyalways true for hypoplastic–anaplastic processes in one or all cell serieswith resulting cytopenia but without hematological signs of malignantcell proliferation.

Procedures, Assays, and Normal Values9Procedures, Assays, and Normal ValuesTaking Blood SamplesSince cell counts are affected by the state of the blood circulation, theconditions under which samples are taken should be the same so far aspossible if comparable values are desired.This means that blood should always be drawn at about the same time ofday and after at least eight hours of fasting, since both circadian rhythmand nutritional status can affect the findings. If strictly comparable valuesare required, there should also be half an hour of bed rest before thesample is drawn, but this is only practicable in a hospital setting. In othersettings (i.e., outpatient clinics), bringing portable instruments to the relaxed,seated patient works well.A sample of capillary blood may be taken when there are no furthertests that would require venous access for a larger sample volume. A wellperfusedfingertip or an earlobe is ideal; in newborns or young infants, theheel is also a good site. If the circulation is poor, the blood flow can be increasedby warming the extremity by immersing it in warm water.Without pressure, the puncture area is swabbed several times with 70%alcohol, and the skin is then punctured firmly but gently with a sterile disposablelancet. The first droplet of blood is discarded because it may becontaminated, and the ensuing blood is drawn into the pipette (seebelow). Care should be taken not to exert pressure on the tissue fromwhich the blood is being drawn, because this too can change the cell compositionof the sample.Obviously, if a venous blood sample is to be taken for the purposes ofother tests, or if an intravenous injection is going to be performed, theblood sample for hematological analysis can be taken from the same site.To do this, the blood is allowed to flow via an intravenous needle into aspecially prepared (commercially available) EDTA-treated tube. The tubeis filled to the 1-ml mark and then carefully shaken several times. The verysmall amount of EDTA in the tube prevents the blood from clotting, butcan itself be safely ignored in the quantitative analysis.

10Physiology and Pathophysiology of Blood CellsErythrocyte CountUp to 20 years ago, blood cells were counted “by hand” in an optical countingchamber. This method has now been almost completely abandoned infavor of automated counters that determine the number of erythrocytesby measuring the impedance or light dispersion of EDTA blood (1 ml), orheparinized capillary blood. Due to differences in the hematocrit, thevalue from a sample taken after (at least 15 minutes’) standing or physicalactivity will be 5–10% higher than the value from a sample taken after 15minutes’ bed rest.Hemoglobin and Hematocrit AssayHemoglobin is oxidized to cyanmethemoglobin by the addition of cyanide,and the cyanmethemoglobin is then determined spectrophotometricallyby the automated counter. The hematocrit describes the ratioof the volume of erythrocytes to the total blood volume (the SI unit iswithout dimension, e.g., 0.4).The EDTA blood is centrifuged in a disposable capillary tube for 10minutes using a high-speed microhematocrit centrifuge (referencemethod). The automated hematology counter determines the mean corpuscularor cell volume (MCV, measured in femtoliters, fl) and the numberof erythrocytes. It calculates the hematocrit (HCT) using the followingformula:HCT = MCV (fl) number of erythrocytes (10 6 /µl).Calculation of Erythrocyte ParametersThe quality of erythrocytes is characterized by their MCV, their mean cellhemoglobin content (MCH), and the mean cellular hemoglobin concentration(MCHC).MCV is measured directly using an automated hemoglobin analyzer, oris calculated as follows:MCV Hematocrit (l/l)Number of erythrocytes (10 6 /µl)MCH (in picograms per erythrocyte) is calculated using the followingformula:Hemoglobin (g/l)MCH (pg) Number of erythrocytes (10 6 /µl)

Procedures, Assays, and Normal Values11MCHC is determined using this formula:MCHC (g/dl) Hemoglobin concentration (g/dl)Hematocrit (l/l)Red Cell Distribution Width (RDW)Modern analyzers also record the red cell distribution width (cell volumedistribution). In normal erythrocyte morphology, this correlates with thePrice-Jones curve for the cell diameter distribution. Discrepancies areused diagnostically and indicate the presence of microspherocytes(smaller cells with lighter central pallor).Reticulocyte CountReticulocytes can be counted using flow cytometry and is based on thelight absorbed by stained aggregates of reticulocyte organelles. The dataare recorded as the number of reticulocytes per mill (‰) of the total numberof erythrocytes. Reticulocytes can, of course, be counted in a countingchamber using a microscope. While this method is not particularlylaborious, it is mostly employed in laboratories that often deal with orhave a special interest in anemia. Reticulocytes are young erythrocytesimmediately after they have extruded their nuclei: they contain, as a remainderof aggregated cell organelles, a net-like structure (hence thename “reticulocyte”) that is not discernible after the usual staining proceduresfor leukocytes, but can be observed after vital staining of cellswith brilliant cresyl blue or new methylene blue. The staining solution ismixed in an Eppendorf tube with an equal volume of EDTA blood and incubatedfor 30 minutes. After repeated mixing, a blood smear is preparedand allowed to dry. The sample is viewed using a microscope equippedwith an oil immersion lens. The ratio of reticulocytes to erythrocytes is determinedand plotted as reticulocytes per 1000 erythrocytes (per mill).Normal values are listed in Table 2, p. 12.

12Physiology and Pathophysiology of Blood CellsTable 2 Normal ranges and mean values for blood cell components*Adults Newborns Toddlers Children 18 years old 1 months 2 years old 10 years oldLeukocytes/µlor 10 6 /l**MV 7000 11000 10000 8000NR 4300–10000Band granulocytes % MV 2 5 3 3NR 0–5Segmented neutrophilic MV 60 30 30 30granulocytesNR 35–85absolute ct./µl MV 3650 3800 3500 4400or 10 6 /l** NR 1850–7250Lymphocytes % MV 30 55 60 40NR 20–50absolute ct./µl MV 2500 6000 6300 3100or 10 6 /l** NR 1500–3500Monocytes % MV 4 6 5 4absolute ct./µl NR 2–6or 10 6 /l** MV 450NR 70–840Eosinophilic granulocytes (%) MV 2 3 2 2NR 0–4absolute ct./µl MV 165or 10 6 /l** NR 0–400

Procedures, Assays, and Normal Values13Basophilic granulocytes (%) MW 0.5 0.5 0.5 0.5NR 0–1Male FemaleErythrocytes 10 6 /µlor 10 12 /l**MV 5.4 4.8 4.7 4.7 4.8NR 4.6–5.9 4.2–5.4 3.9–5.9 3.8–5.4 3.8–5.4Hb g/dlor 10 g/l**MV 15 13 17 12 14NR 14–18 12–16 15–18 11–13 12–15HKT MV 0.45 0.42 44 37 39NR 0.42–0.48 0.38–0.43MCH HbE (pg) MV 29 33 27 25NR 26–32MCV/µm 3or fl**MV 87 91 78 80NR 77–99MCHC g/dlor 10 g/lMV 33 35 33 34NR 33–36Erythrocyte, diameter (µm) MV 7.5 8.1 7.3 7.4Reticulocytes (%) MV 16 24 7.9 7.1 7.6NR 8–25 8–40Thrombocytes 10 3 /µl MV 180 155–566 286–509 247–436NR 140–440MV mean value, NR normal range (range for 95% of the population, reference range), ct. count, ** SI units give the measurements per liter.* For technical reasons, data may vary considerably between laboratories. It is therefore important also to consult the reference ranges of the chosen laboratory.

14Physiology and Pathophysiology of Blood CellsLeukocyte CountLeukocytes, unlike erythrocytes, are completely colorless in their nativestate. Another important physical difference is the stability of leukocytesin 3% acetic acid or saponins; these media hemolyze erythrocytes (thoughnot their nucleated precursors). Türk’s solution, used in most countingmethods, employs glacial acetic acid for hemolysis and crystal violet (gentianviolet) to lightly stain the leukocytes. A 50-µl EDTA blood sample ismixed with 500 µl Türk’s solution in an Eppendorf tube and incubated atroom temperature for 10 minutes. The suspension is again mixed andcarefully transferred to the well of a prepared counting chamber using apipette or capillary tube. The chamber is allowed to fill from a dropletplaced at one edge of the well and placed in a moisture-saturated incubatorfor 10 minutes. With the condenser lowered (or using phase contrastmicroscopy), the leukocytes are then counted in a total of four of the largesquares opposite to each other (1 mm 2 each). The result is multiplied by27.5 (dilution: 1 + 10, volume: 0.4 mm 3 ) to yield the leukocyte count permicroliter. Parallel (control) counts show variation of up to 15%. The normal(reference) ranges are given in Table 2.In an automated blood cell counter, the erythrocytes are lysed and cellswith a volume that exceeds about 30 fl (threshold values vary for differentinstruments) are counted as leukocytes. Any remaining erythroblasts,hard-to-lyse erythrocytes such as target cells, giant thrombocytes, or agglutinatedthrombocytes are counted along with the leukocytes, and thiswill lead to an overestimate of the leukocyte count. Modern analyzers canrecognize such interference factors and apply interference algorithms toobtain a corrected leukocyte count.Visual leukocyte counts using a counting chamber show a variance ofabout 10%; they can be used as a control reference for automatic cellcounts. Rough estimates can also be made by visual assessment of bloodsmears: a 40 objective will show an average of two to three cells per fieldof view if the leukocyte count is normal.

Procedures, Assays, and Normal Values15Thrombocyte CountTo count thrombocytes in a counting chamber, blood must be conditionedwith 2% Novocain–Cl solution. Preprepared commercial tubes are widelyused (e.g., Thrombo Plus with 2-ml content). EDTA blood is pipetted intothe tubes, carefully mixed and immediately placed in a counting chamber.The chamber is allowed to stand for 10 minutes while the cells settle,after which an area of 1 mm 2 is counted. The result corresponds to thenumber of thrombocytes (the 1 + 100 dilution is ignored). In an automatedblood cell counter, the blood cells are counted after they have been sortedby size. Small cells between 2 and 20 fl (thresholds vary for different instruments)are counted as thrombocytes. If giant thrombocytes or agglutinatedthrombocytes are present, they are not counted and the result is anunderestimate. On the other hand, small particles, such as fragmentocytes,or microcytes, will lead to an overestimate. Modern analyzers canrecognize such interference factors and apply interference algorithms toobtain a corrected thrombocyte count. If unexpected results are produced,it is wise to check them by direct reference to the blood smear.Unexpected thrombocyte counts should be verified by direct visualassessment. Using a 100 objective, the field of view normally containsan average of 10 thrombocytes. In some instances, “pseudothrombocytopenias”are found in automated counts. These are artifacts due to thrombocyteaggregation.Pseudothrombocytopenia (see p. 167) is caused by the aggregation ofthrombocytes in the presence of EDTA; it does not occur when heparin orcitrate are used as anticoagulants.Quantitative Normal Values and Range ofCellular Blood ComponentsDetermining normal values for blood components is more difficult andmore risky than one might expect. Obviously, the values are affected by alarge number of variables, such as age, gender, activity (metabolic load),circadian rhythm, and nutrition, not to mention the effects of the bloodsampling technique, type and storage of the blood, and the countingmethod. For this reason, where available, a normal range is given, covering95% of the values found in a clinically normal group of probands—fromwhich it follows that one in every 20 healthy people will have values outsidethe limits of this range. Thus, there are areas of overlap between normaland pathological data. Data in these borderline areas must be interpretedwithin a refined reference range with data from probands who

16Physiology and Pathophysiology of Blood Cellsresemble each other and the patient as closely as possible in respect of thevariables listed above. Due to space limitations, only key age data are consideredhere. Figure 3 clearly shows that, particularly for newborns, toddlers,and young children, particular reference ranges must be taken intoaccount. In addition, the interpretation must also take account ofmethodological variation: in cell counts, the coefficient of variation (standarddeviation as a percentage of the mean value) is usually around 10!22201816Leukocyte count (10 9 /l)1412108642012 2 4 6 1 2 3 5 3 6 9 1 3 5 7 9 11 13LeukocytesGranulocytesLymphocytesMonocytesHoursDaysWeeksMonthsYearsFig. 3 Mean cell counts at different ages in childhood for leukocytes and theirsubfractions (according to Kato)

Procedures, Assays, and Normal Values17In sum, a healthy distrust for the single data point is the most importantbasis for the interpretation of all data, including those outside the referencerange. For every sample of drawn blood, and every countingmethod, at least two or three values should be available before conclusionscan be drawn, unless the clinical findings reflect the cytological data.In addition to this, every laboratory has its own set of reference data tosome extent.After this account of the problems and wide variations between differentgroups, the data in Table 2 are presented in a simplified form, with valuesrounded up or down for ease of comparison and memorization. Absolutevalues and the new SI units are given where they are clinically relevant.The Blood Smear and Its Interpretation(Differential Blood Count, DBC)A blood smear uses capillary or venous EDTA-blood, preferably no morethan three hours old. The slides must be grease-free, otherwise cell aggregationand stain precipitation may occur. Unless commercially availablegrease-free slides are used, the slides should be soaked for several hours ina solution of equal parts of ethanol and ether and then allowed to dry. Adroplet of the blood sample is placed close to the edge of the slide. Aground cover glass (spreader slide) is placed in front of the droplet ontothe slide at an angle of about 30. The cover slide is then slowly backed intothe blood droplet. Upon contact, the blood droplet spreads along the edgeof the slide (Fig. 4). Without pressure, the cover glass is now lightly movedover the slide. The faster the cover glass is moved, and the steeper angle atwhich it is held, the thinner the smear will be.The quality of the smear technique is crucial for the assessment, becausethe cell density at the end of the smear is often twice that at the beginning.In a well-prepared smear the blood sample will show a “feathered” edgewhere the cover glass left the surface of the slide. The smear must bethoroughly air-dried; for good staining, at least two hours’ drying time isneeded. The quality of the preparation will be increased by 10 minutes’fixation with methanol, and it will then also keep better. After drying,name and date are pencilled in on the slide.

18Physiology and Pathophysiology of Blood CellsFig. 4Preparation of a blood smearFor safety’s sake, at least one back-up smear should be made from everysample from every patient.Staining is done with a mixture of basic stains (methylene blue, azure) andacidic stains (eosin), so as to show complementary substances such as nucleicacids and alkaline granulations. In addition to these leukocyte components,erythrocytes also yield different staining patterns: immatureerythrocytes contain larger residual amounts of RNA and therefore stainmore heavily with basophilic stains than do mature erythrocytes. Pappenheim’spanoptic stain contains a balanced mixture of basic and acidicstains: the horizontally stored, air-dried smear is covered with May–Grünwald staining solution (eosin–methylene blue) for three minutes,then about an equal amount of phosphate buffer, pH = 7.3, is carefullyadded and, after a further three minutes, carefully poured off again. Next,the slide is covered with diluted Giemsa stain (azure–eosin), which is preparedby addition of 1 ml Giemsa stock solution to 1 ml neutral distilledwater or phosphate buffer, pH = 6.8–7.4. After 15 minutes, the Giemsastaining solution is gently rinsed off with buffer solution and the smearsare air-dried with the feathered end sloping upwards.The blood smears are initially viewed with a smaller objective (10 to20), which allows the investigator to check the cell density and to findthe best counting area in the smear. Experience shows that the cell projectionis best about 1 cm from the feathered end of the smear. At 40 magnification,one may expect to see an average of two to three leukocytes perviewing field if the leukocyte count is normal. It is sometimes useful to beable to use this rough estimate to crosscheck improbable quantitativevalues. The detailed analysis of the white blood cells is done using an oilimmersion lens and 100 magnification. For this, it is best to scan the sec-

Procedures, Assays, and Normal Values19tion from about 1 cm to about 3 cm from the end of the smear, moving theslide to and fro in a meandering movement across its short diameter.Before (and while) the differential leukocyte count is carried out, erythrocytemorphology and thrombocyte density should be assessed. The resultsof the differential leukocyte count (the morphologies are presented in theatlas section, p. 30 ff.) may be recorded using manual counters or mark-upsystems. The more cells are counted, the more representative the results,so when pathological deviations are found, it is advisable to count 200cells.To speed up the staining process, which can seem long and laboriouswhen a rapid diagnosis is required, several quick-staining sets are availablecommercially, although most of them do not permit comparable fineanalysis. If the standard staining solutions mentioned above are to hand, aquick stain for orientation purposes can be done by incubating the smearwith May–Grünwald reagent for just one minute and shortening theGiemsa incubation time to one to two minutes with concentrated “solution.”Normal values and ranges for the differential blood count are given inTable 2, p. 12.Malaria plasmodia are best determined using a thick smear in additionto the normal blood smear. On a slide, a drop of blood is spread over anarea of about 2.5 cm across. The thick smear is placed in an incubator andallowed to dry for at least 30 minutes. Drying samples as thick smears andthen treating them with dilute Giemsa stain (as described above) achievesextensive hemolysis of the erythrocytes and thus an increase in the releasedplasmodia.Significance of the Automated Blood CountThe qualitative and quantitative blood count techniques described heremay seem somewhat archaic given the now almost ubiquitous automatedcell counters; they are merely intended to show the possibilities alwaysready to be called on in terms of individual analyses carried out by small,dedicated laboratories.The automated cell count has certainly rationalized blood cell counting.Depending on the diagnostic problem and the quality control system ofthe individual laboratory, automated counting can even reduce dataranges compared with “manual” counts.After lysis of the erythrocytes, hematology analyzers determine the numberof remaining nucleated cells using a wide range of technologies. Allcounters use cell properties such as size, interaction with scattered light atdifferent angles, electrical conductivity, nucleus-to-cytoplasm ratio, and

20Physiology and Pathophysiology of Blood Cellsthe peroxidase reaction, to group individual cell impulses into clusters.These clusters are then quantified and assigned to leukocyte populations.If only normal blood cells are present, the assignment of the clusters tothe various leukocyte populations works well, and the precision of the automatedcount exceeds that of the manual count of 100 cells in a smear bya factor of 10. If large number of pathological cells are present, such asblasts or lymphadenoma cells, samples are reliably recognized as “pathological,”and a smear can then be prepared and further analyzed under themicroscope.The difficulty arises when small populations of pathological cells arepresent (e.g., 2% blasts present after chemotherapy), or when pathologicalcells are present that closely resemble normal leukocytes (e.g., small centrocytesin satellite cell lymphoma). These pathological conditions are notalways picked up by automated analyzers (false negative result), no smearis prepared and studied under the microscope, and the results producedby the machine do not include the presence of these pathological populations.For this reason, blood samples accompanied by appropriate clinicalqueries (e.g., “lymphadenoma?” “blasts?” “unexplained anemia?”) shouldalways be differentiated and evaluated using a microscope.Bone Marrow BiopsyOccasionally, a disease of the blood cell system cannot be diagnosed andclassified on the basis of the blood count alone and a bone marrow biopsyis indicated. In such cases it is more important to perform this biopsy competentlyand produce good smears for evaluation than to be able to interpretthe bone marrow cytology yourself. Indications for bone marrowbiopsy are given in Table 3.In the attempt to avoid complications, the traditional location for bonemarrow biopsy at the sternum has been abandoned in favor of the superiorpart of the posterior iliac spine (back of the hipbone) (Fig. 5).Although the bone marrow cytology findings from the aspirate are sufficientor even preferable for most hematological questions (see Table 3), itis regarded as good practice to obtain a sample for bone marrow histologyat the same time, since with improved instruments the procedurehas become less stressful, and complementary cytological and histologicaldata are then available from the start. After deep local anesthesia of thedorsal spine and a small skin incision, a histology cylinder at least 1.5 cmlong is obtained using a sharp hollow needle (Yamshidi). A Klima and Rosseggercytology needle (Fig. 5) is then placed through the same subcutaneouschannel but at a slightly different site from the earlier insertionpoint on the spine and gently pushed through the compacta. The mandrel

Procedures, Assays, and Normal Values21is pulled out and a 5- to 10-ml syringe body with 0.5 ml citrate or EDTA(heparin is used only for cytogenetics) is attached to the needle. Thepatient should be warned that there will be a painful drawing sensationduring aspiration, which cannot be avoided. The barrel is then slowlypulled, and if the procedure is successful, blood from the bone marrow fillsthe syringe. The syringe body is separated from the needle and the mandrelreintroduced. The bone marrow aspirate is transferred from the syringeto a Petri dish. When the dish is gently shaken, small, pinhead-sizedbone marrow spicules will be seen lying on the bottom. A smear, similar toa blood smear, can be prepared on a slide directly from the remaining contentsof the syringe. If the first aspirate has obtained material, the needle isremoved and a light compression bandage is applied.If the aspirate for cytology contains no bone marrow fragments (“punctiosicca,” dry tap), an attempt may be made to obtain a cytology smearfrom the (as yet unfixed) histology cylinder by rolling it carefully on theslide, but this seldom produces optimal results.The preparation of the precious bone marrow material demandsspecial care. One or two bone marrow spicules are pushed to the outeredge of the Petri dish, using the mandrel from the sternal needle, a needle,or a wooden rod with a beveled tip, and transferred to a fat-free microscopyslide, on which they are gently pushed to and fro by the needle alongthe length of the slide in a meandering line. This helps the analyzingFig. 5 Bone marrow biopsy from the superior part of the posterior iliac spine(back of the hipbone)

22Physiology and Pathophysiology of Blood CellsFig. 6 Squash preparation and meandering smear for the cytological analysis ofbone marrow spiculestechnician to make a differentiatial count. It should be noted that toomuch blood in the bone marrow sample will impede the semiquantitativeanalysis. In addition to this type of smear, squash preparations should alsobe prepared from the bone marrow material for selective staining. To dothis, a few small pieces of bone marrow are placed on a slide and coveredby a second slide. The two slides are lightly pressed and slid against eachother, then separated (see Fig. 6).The smears are allowed to air-dry and some are incubated with panopticPappenheim staining solution (see previous text). Smears being sent toa diagnostic laboratory (wrapped individually and shipped as fragilegoods) are better left unstained. Fresh smears of peripheral blood shouldaccompany the shipment of each set of samples. (For principles of analysisand normal values see p. 52 ff., for indications for bone marrow cytologyand histology see p. 27 ff.)

Procedures, Assays, and Normal Values23Lymph Node Biopsy and Tumor BiopsyThese procedures, less invasive than bone marrow biopsy, are a simpleand often diagnostically sufficient method for lymph node enlargement orother intumescences. The unanesthetized, disinfected skin is sterilizedand pulled taut over the node. A no. 1 needle on a syringe with good suctionis pushed through the skin into the lymph node tissue (Fig. 7). Tissueis aspirated from several locations, changing the angle of the needleslightly after each collection, and suction maintained while the needle iswithdrawn into the subcutis. Aspiration ceases and the syringe is removedwithout suction. The biopsy harvest, which is in the needle, is extrudedonto a microscopy slide and smeared out without force or pressure using acover glass (spreader slide). Staining is done as described previously forblood smears.

24Physiology and Pathophysiology of Blood Cells1.Skin puncture2.Aspiration3.Collectingaspirates fromdifferent lymphnode locations4.Detaching thesyringe body,equalizing thepressure difference5.Removal of thesyringe body andcannula6.Pulling back thesyringe barrel7.Pushing thebiopsy materialonto a slideFig. 7 Procedure forlymph node biopsy

Step-by-Step Diagnostic Sequence25Step-by-Step Diagnostic SequenceOn the basis of what has been said so far, the following guidelines for thediagnostic workup of hematological changes may be formulated:1. The first step is quantitative determination of leukocytes (L), erythrocytes(E) and thrombocytes (T). Because the normal range can vary so widelyin individual cases (Table 2), the following rule of thumb should be observed:A complete blood count (CBC) should be included in the baseline data,like blood pressure.2. All quantitative changes in L + E + T call for a careful evaluation of thedifferential blood count (DBC). Since clinical findings determinewhether a DBC is indicated, it may be said that:A differential blood count is indicated:➤ By all unexplained clinical symptoms, especially➤ Enlarged lymph nodes or splenomegaly➤ Significant changes in any of– Hemoglobin content or number of erythrocytes– Leukocyte count– Thrombocyte countThe only initial assumption here is that mononuclear cells with unsegmentednuclei can be distinguished from polynuclear cells. While thisnomenclature may not conform to ideal standards, it is well establishedand, moreover, of such practical importance as a fundamental distinguishingcriterion that it is worth retaining. A marked majority ofmononuclear cells over the polynuclear segmented granulocytes is anunambiguous early finding.The next step has to be taken with far more care and discernment:classifying the mononuclear cells according to their possible origin, lymphaticcells, monocytes, or various immature blastic elements, whichotherwise only occur in bone marrow. The aim of the images in theAtlas section of this book is to facilitate this part of the differential diagnosis.To a great extent, the possible origins of mononuclear cells can bedistinguished; however, the limits of morphology and the vulnerabilityto artifacts are also apparent, leaving the door wide open to further

26Physiology and Pathophysiology of Blood Cellsdiagnostic steps (specialist morphological studies, cytochemistry,immunocytochemistry. A predominance of mononuclear cell elementshas the same critical significance in the differential diagnosis of bothleukocytoses and leukopenias.3. After the evaluation of the leukocytes, assessment of erythrocyte morphologyis a necessary part of every blood smear evaluation. It is, naturally,particularly important in cases showing disturbances in theerythrocyte count or the hemoglobin.4. After careful consideration of the results obtained so far and thepatient's clinical record, the last step is the analysis of the cell compositionof the bone marrow. Quite often, suspected diagnoses are confirmedthrough humoral tests such as electrophoresis, or through cytochemicaltests such as alkaline phosphatase, myeloperoxidase, nonspecificesterase, esterase, or iron tests.Bone marrow analysis is indicated when clinical findings and bloodanalysis leave doubts in the diagnostic assessment, for example incases of:➤ Leukocytopenia➤ Thrombocytopenia➤ Undefined anemia➤ Tricytopenia, or➤ Monoclonal hypergammaglobulinemiaA bone marrow analysis may be indicated in order to evaluate thespreading of a lymphadenoma or tumor, unless the bloodstream alreadyshows the presence of pathological cells.5. Bone marrow histology is also rarely indicated (even more rarely thanthe bone marrow cytology). Examples of the decision-making processbetween bone marrow cytology and histology (biopsy) are shown inTable 3.Often only histological analysis can show structural changes or focal infiltrationof the bone marrow.This is particularly true of the frequently fiber-rich chronic myeloproliferativediseases, such as polycythemia vera rubra, myelofibrosis–osteomyelosclerosis (MF-OMS), essential thrombocythemia (ET), andchronic myeloid leukemia (CML) as well as malignant lymphomawithout hematological involvement (Hodgkin disease or blastic non-Hodgkin lymphoma) and tumor infiltration.

Step-by-Step Diagnostic Sequence27Table 3Indications for a differential blood count (DBC), bone marrow aspiration, and biopsyIndicationsAll clinically unclear situations:➤ Enlarged lymph nodes or spleen➤ Changes in the simple CBC (penias orcytoses)➤ When a diagnosis cannot be made based onclinical findings and analysis of peripheralblood differential blood analysis, cytochemistry,phenotyping, molecular genetics orFISHProceduresDifferential blood count (DBC)Bone marrow analysisBone marrow aspirate(Morphology, phenotyping,cytogenetics, FISH,molecular genetics)Bone marrowtrephine biopsy(Morphology, immunohistology)➤ Punctio sicca ("dry tap") not possible +➤ Aplastic bone marrow not possible +➤ Suspicion of myelodysplastic syndrome + (+) (e. g. hypoplasia)➤ Pancytopenia + +➤ Anemia, isolated + –➤ Granulocytopenia, isolated + –➤ Thrombocytopenia, isolated (except ITP) + –➤ Suspected ITP (+) For therapy failure –➤ Suspected OMF/OMS – (BCR-ABL in peripheral +blood is sufficient)➤ Suspected PV – (BCR-ABL in peripheral –blood is sufficient)➤ Suspected ET – (BCR-ABL in peripheral +blood is sufficient)➤ Suspected CML – (BCR-ABL in peripheralblood is sufficient)(+) Conditions for theanalysis➤ CMPE (BCR-ABL negative) (+) Differential diagnoses +➤ Suspected AL/AL + (+) Not in typical cases➤ Suspected bone marrow metastases – +➤ Monoclonal hypergammaglobulinemia + +➤ NHL (exceptions, see below) + +➤ Typical CLL + (+) Prognostic factor➤ Follicular lymphoma (+) To distinguish vs +other NHL➤ Hodgkin disease – +Further indications for a bone marrow analysis are:➤ Unexplained hypercalcemia + +➤ Inexplicable increase of bone AP – +➤ Obvious, unexplained abnormalities – +➤ Hyperparathyroidism – +➤ Paget disease – +➤ Osteomalacia – +➤ Renal osteopathy – +➤ Gaucher syndrome – ++ Recommended, – not recommended, (+) conditionally recommended, ITP idiopathic thrombocytopenia,OMF/OMS osteomyelofibrosis/osteomyelosclerosis, PV polycythemia vera, ET essential thrombocythemia,CMPD chronic myeloproliferative disorders, AL acute leukemia, NHL non-Hodgkin lymphoma, CLL chroniclymphocytic leukemia, FISH fluorescence in situ hybridization, AP alkaline phosphatase.

Normal Cells of the Blood andHematopoietic Organs

30Normal Cells of the Blood and Hematopoietic OrgansThe Individual Cells of HematopoiesisImmature Red Cell Precursors: Proerythroblasts andBasophilic ErythroblastsProerythroblasts are the earliest, least mature cells in the erythrocyteformingseries (erythropoiesis). Proerythroblasts are characterized bytheir size (about 20 µm), and by having a very dense nuclear structurewith a narrow layer of cytoplasm, homogeneous in appearance, with alighter zone at the center; they stain deep blue after Romanowsky staining.These attributes allow proerythroblasts to be distinguished frommyeloblasts (p. 35) and thus to be assigned to the erythrocyte series. Aftermitosis, their daughter cells display similar characteristics except thatthey have smaller nuclei. Daughter cells are called basophilic erythroblasts(formerly also called macroblasts). Their nuclei are smaller and the chromatinis more coarsely structured.The maturation of cells in the erythrocyte series is closely linked to theactivity of macrophages (transformed monocytes), which phagocytosenuclei expelled from normoblasts and iron from senescent erythrocytes,and pass these cell components on to developing erythrocytes.Diagnostic Implications. Proerythrocytes exist in circulating blood onlyunder pathological conditions (extramedullary hematopoiesis; breakdownof the blood–bone marrow barrier by tumor metastases, p. 150; orerythroleukemia, p. 100). In these situations, basophilic erythroblasts mayalso occur; only exceptionally in the course of a strong postanemia regenerationwill a very few of these be released into the blood stream (e.g.,in the compensation phase after severe hemorrhage or as a response tovitamin deficiency, see p. 152).

Normally erythropoiesis takes place only in the bone marrowabcFig. 8 Early erythropoiesis. a The earliest recognizable red cell precursor is thelarge dark proerythroblast with loosely arranged nuclear chromatin (1). Below aretwo orthochromatic erythroblasts (2), on the right a metamyelocyte (3). b Proerythroblast(1). c Proerythroblast (1) next to a myeloblast (2) (see p. 34); lowerregion of image shows a promyelocyte (3). Toward the upper left are a metamyelocyte(4) and a segmented neutrophilic granulocyte (5).31

During increased turnover, nucleated red cell precursors maymigrate into the peripheral bloodabcdeFig. 9 d The density of the nuclear chromatin is similar in lymphocytes (1) anderythroblasts (2), but in the erythroblast the cytoplasm is wider and similar in colorto a polychromatic erythrocyte (3). e Normal red blood cell findings with slightvariance in size of the erythrocytes. A lymphocyte (1) and a few thrombocytes (2)are seen. The erythrocytes are slightly smaller than the nucleus of the lymphocytenucleus.33

34Normal Cells of the Blood and Hematopoietic OrgansImmature White Cell Precursors: Myeloblasts andPromyelocytesMyeloblasts are the least mature cells in the granulocyte lineage. Mononuclear,round-to-ovoid cells, they may be distinguished from proerythroblastsby the finer, “grainy” reticular structure of their nuclei and thefaintly basophilic cytoplasm. On first impression, they may look like largeor even small lymphocytes (micromyeloblasts), but the delicate structureof their nuclei always gives them away as myeloblasts. In some areas, condensedchromatin may start to look like nucleoli. Sporadically, the cytoplasmcontains azurophilic granules.Promyelocytes are the product of myeloblast division, and usually growlarger than their progenitor cells. During maturation, their nuclei show anincreasingly coarse chromatin structure. The nucleus is eccentric; thelighter zone over its bay-like indentation corresponds to the Golgi apparatus.The wide layer of basophilic cytoplasm contains copious largeazurophilic granules containing peroxidases, hydrolases, and otherenzymes. These granulations also exist scattered all around the nucleus, asmay be seen by focusing on different planes of the preparation using themicrometer adjustment on the microscope.Diagnostic Implications. Ordinarily, both cell types are encountered onlyin the bone marrow, where they are the most actively dividing cells andmain progenitors of granulocytes. In times of increased granulocyte production,promyelocytes and (in rare cases) myeloblasts may be releasedinto the blood stream (pathological left shift, see p. 112). Under strong regenerationpressure from the erythrocyte series, too—e.g., during thecompensation phase following various anemias—immature white cellprecursors, like the red cell precursors, may be swept into the peripheralblood. Bone marrow involvement by tumor metastases also increases thepermeability of the blood–bone marrow barrier for immature white cellprecursors (for an overview, see p. 112 ff.).In some acute forms of leukemia, myeloblasts (and also, rarely, promyelocytes)dominate the blood analysis (p. 97).

Round cells with “grainy” reticular chromatin structure areblasts, not lymphocytesabcdFig. 10 Granulocyte precursors. a The least mature precursor in granulopoiesisis the myeloblast, which is released into the blood stream only under pathologicalconditions. A large myeloblast is shown with a fine reticular nuclear structure anda narrow layer of slightly basophilic cytoplasm without granules. b Myeloblast andneutrophilic granulocytes with segmented nuclei (blood smear from a patientwith AML). c Myeloblast (1), which shows the start of azurophilic granulation(arrow), and a promyelocyte (2) with copious large azurophilic granules, typicallyin a perinuclear location. d Large promyelocyte (1), myelocyte (2), metamyelocyte(3), and polychromatic erythroblast (4).35

36Normal Cells of the Blood and Hematopoietic OrgansPartly Mature White Cell Precursors: Myelocytes andMetamyelocytesMyelocytes are the direct product of promyelocyte mitosis and are alwaysclearly smaller than their progenitors. The ovoid nuclei have a bandedstructure; the cytoplasm is becoming lighter with maturation and in somecases acquiring a pink tinge. A special type of granules, which no longerstain red like the granules in promyelocytes (“specific granules,” peroxidase-negative),are evenly distributed in the cytoplasm. Myelocyte morphologyis wide-ranging because myelocytes actually cover three differentvarieties of dividing cells.Metamyelocytes (young granulocytes) are the product of the final myelocytedivision and show further maturation of the nucleus with an increasingnumber of stripes and points of density that give the nuclei a spottedappearance. The nuclei slowly take on a kidney bean shape and have someplasticity. Metamyelocytes are unable to divide. From this stage on, onlyfurther maturation of the nucleus occurs by contraction, so that the distinctions(between metamyelocytes, band neutrophils, and segmentedneutrophils) are merely conventional, although they do relate to the varying“maturation” of these cell forms.Diagnostic Implications. Like their precursors, myelocytes and metamyelocytesnormally appear in the peripheral blood only during increasedcell production in response to stress or triggers, especially infections (foran overview of possible triggers, see p. 112). Under these conditions, theyare, however, more abundant than myeloblasts or promyelocytes.

Myelocytes and metamyelocytes also occur in the blood streamin severe reactive diseaseabcdFig 11 Myelocytes and metamyelocytes. a Early myelocyte. The chromatinstructure is denser than that of promyelocytes. The granules do not lie over thenucleus (as can be seen by turning the fine focus adjustment of the microscope toand fro). The blood smear is from a case of sepsis, hence the intensive granulation.b Slightly activated myelocyte (the cytoplasm is still relatively basophilic). c Typicalmyelocyte (1) close to a segmented neutrophil (2). d This metamyelocyte isdistinguished from a myelocyte by incipient lobe formation.37

Advancing nuclear contraction and segmentation: continuoustransformation from metamyelocyte to band cell and then segmentedneutrophilic granulocyteabcdfegFig. 12 Neutrophils (neutrophilic granulocytes). a Transitional form between ametamyelocyte and a band cell. b Copious granulation in a band cell (1) (toxic granulation)next to band cells (2) with Döhle bodies (arrows). c Two band cells.d Band cells can also occur as aggregates. e Segmented neutrophilic granulocytes.f Segmented neutrophilic granulocyte after the peroxidase reaction.g Segmented neutrophilic granulocyte after alkaline leukocyte phosphatase (ALP)staining.39

40Normal Cells of the Blood and Hematopoietic OrgansCell Degradation, Special Granulations, and NuclearAppendages in Neutrophilic Granulocytes and NuclearAnomaliesToxic granulation is the term used when the normally faint stippledgranules in segmented neutrophils stain an intense reddish violet, usuallyagainst a background of slightly basophilic cytoplasm; unlike the normalgranules, they stain particularly well in an acidic pH (5.4). This phenomenonis a consequence of activity against bacteria or proteins and is observedin serious infections, toxic or drug effects, or autoimmuneprocesses (e.g., chronic polyarthritis). At the same time, cytoplasmicvacuoles are often found, representing the end stage of phagocytosis (especiallyin cases of sepsis), as are Döhle bodies: small round bodies of basophiliccytoplasm that have been described particularly in scarlet fever,but may be present in all serious infections and toxic conditions. A deficiencyor complete absence of granulation in neutrophils is a sign ofsevere disturbance of the maturation process (e.g., in myelodysplasia oracute leukemia). The Pelger anomaly, named after its first describer, is ahereditary segmentation anomaly of granulocytes that results in round,rod-shaped, or bisegmented nuclei. The same appearance as a nonhereditarycondition (pseudo-Pelger formation, also called Pel–Ebstein fever, or[cyclic] Murchison syndrome) indicates a severe infectious or toxic stressresponse or incipient myelodysplasia; it also may accompany manifestleukemia.

Note the granulations, inclusions, and appendages in segmentedneutrophilic granulocytesabcFig. 13dVariations of segmented neutrophilic granulocytes. a Reactive state withtoxic granulation of the neutrophilic granulocytes, more visibly expressed in thecell on the left (1) than the cell on the right (2) (compare with nonactivated cells,p. 39). b Sepsis with toxic granulation, cytoplasmic vacuoles, and Döhle bodies(arrows) in band cells (1) and a monocyte (2). c Pseudo-Pelger cell looking likesunglasses (toxic or myelodysplastic cause). d Döhle-like basophilic inclusion (arrow)without toxic granulation. Together with giant thrombocytes this suggestsMay–Hegglin anomaly.continued 41

Note the granulations, inclusions, and appendages in segmentedneutrophilic granulocytesefghFig. 13 continued. e Hypersegmented neutrophilic granulocyte (six or moresegments). There is an accumulation of these cells in megaloblastic anemia. fDrumstick (arrow 1) as an appendage with a thin filament bridge to the nucleus(associated with the X-chromosome), adjoined by a thrombocyte (arrow 2). gVery large granulocyte from a blood sample taken after chemotherapy. h Segmentedneutrophilic granulocyte during degradation, often seen as an artifact afterprolonged sample storage (more than eight hours).43

44Normal Cells of the Blood and Hematopoietic OrgansEosinophilic Granulocytes (Eosinophils)Eosinophils arise from the same stem cell population as neutrophils andmature in parallel with them. The earliest point at which eosinophils canbe morphologically defined in the bone marrow is at the promyelocytestage. Promyelocytes contain large granules that stain blue–red; not untilthey reach the metamyelocyte stage do these become a dense populationof increasingly round, golden-red granules filling the cytoplasm. TheCharcot–Leyden crystals found between groups of eosinophils in exudatesand secretions have the same chemical composition as the eosinophilgranules.The nuclei of mature eosinophils usually have only two segments.Diagnostic Implications. In line with their function (see p. 5) (reactionagainst parasites and regulation of the immune response, especiallydefense against foreign proteins), an increase of eosinophils above 400/µlshould be seen as indicating the presence of parasitosis, allergies, andmany other conditions (p. 124).Basophilic Granulocytes (Basophils)Like eosinophils, basophils (basophilic granulocytes) mature in parallelwith cells of the neutrophil lineage. The earliest stage at which they can beidentified is the promyelocyte stage, at which large, black–violet stainedgranules are visible. In mature basophils, which are relatively small, thesegranules often overlie the two compact nuclear segments like blackberries.However, they easily dissolve in water, leaving behind faintly pinkstained vacuoles.Close relations of basophilic granulocytes are tissue basophils or tissuemast cells—but these are never found in blood. Tissue basophils have around nucleus underneath large basophilic granules.Diagnostic Implications. In line with their role in anaphylactic reactions(p. 5), elevated basophil counts are seen above all in hypersensitivity reactionsof various kinds. Basophils are also increased in chronic myeloproliferativebone marrow diseases, especially chronic myeloid leukemia(pp. 117, 120).

Round granules filling the cytoplasm: eosinophilic and basophilicgranulocytesabcdefFig. 14 Eosinophilic and basophilic granulocytes. a–c Eosinophilic granulocyteswith corpuscular, orange-stained granules. d In contrast, the granules of neutrophilicgranulocytes are not round but more bud-shaped. e Basophilic granulocyte.The granules are corpuscular like those of the eosinophilic granulocyte but staindeep blue to violet. f Very prominent large granules in a basophilic granulocyte inchronic myeloproliferative disease.45

46Normal Cells of the Blood and Hematopoietic OrgansMonocytesMonocytes are produced in the bone marrow; their line of developmentbranches off at a very early stage from that of the granulocytic series (seeflow chart p. 3), but does not contain any distinct, specific precursors thatcan be securely identified with diagnostic significance in everyday morphologicalstudies. Owing to their great motility and adhesiveness, maturemonocytes are morphologically probably the most diversified of allcells. Measuring anywhere between 20 and 40 µm in size, their constantcharacteristic is an ovoid nucleus, usually irregular in outline, with invaginationsand often pseudopodia-like cytoplasmic processes. The fine,“busy” structure of their nuclear chromatin allows them to be distinguishedfrom myelocytes, whose chromatin has a patchy, streaky structure,and also from lymphocytes, which have dense, homogeneous nuclei.The basophilic cytoplasmic layer varies in width, stains a grayish color,and contains a scattered population of very fine reddish granules that areat the very limit of the eye’s resolution. These characteristics vary greatlywith the size of the monocyte, which in turn is dependent on the thicknessof the smear. Where the smear is thin, especially at the feathered end,monocytes are abundant, relatively large and loosely structured, and theircytoplasm stains light gray–blue (“dove gray”). In thick, dense parts of thesmear, some monocytes look more like lymphocytes: only a certain nuclearindentation and the “thundercloud” gray–blue staining of the cytoplasmmay still mark them out.Diagnostic Implications. In line with their function (see p. 5) as phagocyticdefense cells, an elevation of the monocyte population above 7% andabove 850/µl indicates an immune defense reaction; only when a sharprise in monocyte counts is accompanied by a drop in absolute counts inthe other cell series is monocytic leukemia suggested (p. 101). Phagocytosisof erythrocytes and white blood cells (hemophagocytosis) may occurin some virus infections and autoimmune diseases.➤ Monocytosis in cases of infection: always present at the end of acute infections;chronic especially in– Endocarditis lenta, listeriosis, brucellosis, tuberculosis➤ Monocytosis in cases of a non-infectious response, e.g.,– Collagenosis, Crohn disease, ulcerative colitis➤ Monocytoses in/as neoplasia, e.g.,– Paraneoplastic in cases of disseminating tumors, bronchial carcinoma,breast carcinoma, Hodgkin disease, myelodysplasias (especiallyCMML, pp. 107 f) and acute monocytic leukemia (p. 101)

Monocytes show the greatest morphological variation amongblood cellsabcdefghFig. 15 Monocytes. a–c Range of appearances of typical monocytes with lobed,nucleus, gray–blue stained cytoplasm and fine granulation. d Phagocytic monocytewith plasma vacuoles. e Monocyte (1) to the right of a lymphocyte with azurophilicgranules (2). f Monocyte (1) with nucleus resembling that of a band neutrophil,but its cytoplasm stains typically gray–blue. Lymphocyte (2). g A monocytethat has phagocytosed two erythrocytes and harbors them in its wide cytoplasm(arrows) (sample taken after bone marrow transplantation). h Esterasestaining, a typical marker enzyme for cells of the monocyte lineage.47

48Normal Cells of the Blood and Hematopoietic OrgansLymphocytes (and Plasma Cells)Lymphocytes are produced everywhere, particularly in the lymph nodes,spleen, bone marrow, and the lymphatic islands of the intestinal mucosa,under the influence of the thymus (T-lymphocytes, about 80%), or thebone marrow (B-lymphocytes, about 20%). A small fraction of the lymphocytesare NK cells (natural killer cells). Immature precursor cells (lymphnode cytology, p. 177) are practically never released into the blood and aretherefore of no practical diagnostic significance. The cells encountered incirculating blood are mostly “small” lymphocytes with oval or round nuclei6–9 µm in diameter. Their chromatin may be described as dense andcoarse. Detailed analysis under the microscope, using the micrometerscrew to view the chromatin in different planes, reveals not the patch-likeor banded structure of myeloblast chromatin, or the “busy” structure ofmonocyte chromatin, but slate-like formations with homogeneous chromatinand intermittent narrow, lighter layers that resemble geologicalbreak lines. Nucleoli are rarely seen. The cytoplasm wraps quite closelyaround the nucleus and is slightly basophilic. Only a few lymphocytes displaythe violet stained stippling of granules; about 5% of small lymphocytesand about 3% of large ones. The family of large lymphocytes withgranulation consists mostly of NK cells. An important point is that smalllymphocytes—which cannot be identified as T- or B-lymphocytes on thebasis of morphology—are not functional end forms, but undergo transformationin response to specific immunological stimuli. The final stage of B-lymphocyte maturation (in bone marrow and lymph nodes) is plasmacells, whose nuclei often show radial bars, and whose basophilic cytoplasmlayer is always wide. Intermediate forms (“plasmacytoid” lymphocytes)also exist.Diagnostic Implications. Values between 1500 and 4000/µl and about 35%reflect normal output of the lymphatic system. Elevated absolute lymphocytecounts, often along with cell transformation, are observed predominantlyin viral infections (pp. 67, 69) or in diseases of the lymphatic system(p. 75 ff.). Relative increases at the expense of other blood cell series maybe a manifestation of toxic or aplastic processes (agranulocytosis, p. 87;aplastic anemia, p. 148), because these irregularities are rare in the lymphaticseries. A spontaneous decrease in lymphocyte counts is normallyseen only in very rare congenital diseases (agammaglobulinemia [Brutondisease], DiGeorge disease [chromosome 22q11 deletion syndrome]).Some systemic diseases also lead to low lymphocyte counts (Hodgkin disease,active AIDS).Mature plasma cells are rarely found in blood (plasma cell leukemia isextremely rare). Plasma-cell-like (“plasmacytoid”) lymphocytes occur inviral infections or systemic diseases (see p. 68 f. and p. 74 f.).

Lymphocytes are small round cells with dense nuclei and somevariation in their appearanceabcdefgFig. 16 Lymphocytes a–c Range of appearance of normal lymphocytes (some ofthem adjacent to segmented neutrophilic granulocytes). d In neonates, somelymphocytes from a neonate show irregularly shaped nuclei with notches or hintsof segmentation. e A few larger lymphocytes with granules may occur in a normalperson. f Occasionally, and without any recognizable trigger, the cytoplasm maywiden. g A smear taken after infection may contain a few plasma cells, the final,morphologically fully developed cells in the B-lymphocyte series (for further activatedlymphocyte forms, see p. 67).49

50Normal Cells of the Blood and Hematopoietic OrgansMegakaryocytes and ThrombocytesMegakaryocytes can enter the bloodstream only in highly pathologicalmyeloproliferative disease or acute leukemia. They are shown here inorder to demonstrate thrombocyte differentiation. Megakaryocytes residein the bone marrow and have giant, extremely hyperploid nuclei (16times the normal number of chromosome sets on average), which build upby endomitosis. Humoral factors regulate the increase of megakaryocytesand the release of thrombocytes when more are needed (e.g., bleeding orincreased thrombocyte degradation). Cytoplasm with granules is pinchedoff from megakaryocytes to form thrombocytes. The residual naked megakaryocytenuclei are phagocytosed.Only mature thrombocytes occur in blood. About 1–4 µm in size andanuclear, their light blue stained cytoplasm and its processes give them astar-like appearance, with fine reddish blue granules near the center.Young thrombocytes are larger and more “spread out;” older ones looklike pyknotic dots.Diagnostic Implications. In a blood smear, there are normally 8–15 thrombocytesper view field using a 100 objective; they may appear dispersedor in groups. To someone quickly screening a smear, they will give a goodindication of any increase or strong decrease in the count, which can beuseful for early diagnosis of acute thrombocytopenias (p. 164 f.).Small megakaryocyte nuclei are found in the bloodstream only insevere myeloproliferative disorders (p. 171).

Megakaryocytes are never present in a normal peripheral bloodsmear. Thrombocytes are seen in every field viewabcdFig. 17 Megakaryocytes and thrombocytes. a Megakaryocytes in a bone marrowsmear. The wide cytoplasm displays fine, cloudy granulation as a sign of incipientthrombocyte budding. b Normal density of thrombocytes among the erythrocytes,with little variation in thrombocyte size. c and d Peripheral blood smearswith aggregations of thrombocytes. When such aggregates are seen against abackground of apparent thrombocytopenia, the phenomenon is called “pseudothrombocytopenia”and is usually an effect of the anticoagulant EDTA (see alsop. 167).51

52Normal Cells of the Blood and Hematopoietic OrgansBone Marrow: Cell Compositionand Principles of AnalysisAs indicated above, and as will be shown below, almost all disorders of thehematopoietic system can be diagnosed using clinical findings, bloodanalysis, and humoral data. There is no mystery about bone marrow diagnostics.The basic categories are summarized here to give an understandingof how specific diagnostic information is achieved; photomicrographswill show the appearance of specific diseases. Once the individual celltypes, as given in the preceding pages, are recognized, it becomes possibleto interpret the bone marrow smears that accompany the various diseases,and to follow further, analogous diagnostic steps. As a first step inthe analysis of a bone marrow tissue smear or squash preparation, variousareas in several preparations are broadly surveyed. This is followed by individualanalysis of at least 200 cells from two representative areas. Table4 shows the mean normal values and their wide ranges.A combination of estimation and quantitative analysis is used, based onthe following criteria:Cell Density. This parameter is very susceptible to artifacts. Figure 18shows roughly the normal cell density. A lower count may be due to themanner in which the sample was obtained or to the smearing procedure. Abone marrow smear typically shows areas where connective tissue adipocyteswith large vacuoles predominate. Only if these adipocytes areas arepresent is it safe to assume that the smear contains bone marrow materialand that an apparent deficit of bone marrow cells is real.Increased cell density: e.g., in all strong regeneration or compensationprocesses, and in cases of leukemia and myeloproliferative syndromes(except osteomyelosclerosis).Decreased cell density: e.g., in aplastic processes and myelofibrosis.

Bone Marrow: Cell Composition and Principles of Analysis53Table 4 Cell composition in the bone marrow: normal values (%)Medianvalues(J. Boll)Median values and normalrange(K. Rohr)Red cell series– Proerythrocytes 1– Macroblasts (basophilic 3 3.5 0.5–7.5erythroblasts)– Normoblasts (poly- andorthochromic erythroblasts)16 19 (7–40)Neutrophil series– Myeloblasts 2.7 1 (0.5–5)– Promyelocytes 9.5 3 (0–7.5)– Myelocytes 14 15 (5–25)– Metamyelocytes 10.5 15 (5–20)– Band neutrophils 9.8 15 (5–25)– Segmented neutrophils 17.5 7 (0.5–15)Small cell series– Eosinophilic granulocytes 5 3 (1–7)– Basophilic granulocytes 1 0.5 (0–1)– Monocytes 2 2 (0.5–3)– Lymphocytes 6 7.5 (2.5–15)– Plasma cells 1.5 1 (0.5–3)MegakaryocytesCell densities vary widely, 0.5–2 per view field during screening at low magnification.Ratios of Red Cell Series to White Cell Series. In the final analysis, bonemarrow cytology allows a quantitative assessment only in relative terms.The important ratio of red precursor cells to white cells is 1 : 2 for menand 1 : 3 for women.Shifts towards erythropoiesis are seen in all regenerative anemias (hemorrhagicanemia, iron deficiency anemia, vitamin deficiency anemia, andhemolysis), pseudopolycythemia (Gaisböck syndrome), and polycythemia,also in rare pseudo-regenerative disorders, such as sideroachresticanemia and myelodysplasias. Shifts toward granulopoiesis are seenin all reactive processes (infections, tumor defense) and in all malignantprocesses of the white cell series (chronic myeloid leukemia, acuteleukoses).

54Normal Cells of the Blood and Hematopoietic OrgansDistribution and Cell Quality in Erythropoiesis. In erythropoiesis polychromaticerythroblasts normally predominate. Proerythroblasts and basophilicerythroblasts only make up a small portion (Table 4). Here, too, aleft shift indicates an increase in immature cell types and a right shift anincrease in orthochromatic erythroblasts. Qualitatively, vitamin B 12 andfolic acid deficiency lead to a typical loosening-up of the nuclear structurein proerythroblasts and to nuclear segmentation and break-up in theerythroblasts (megaloblastic erythropoiesis).A left shift is seen in regenerative anemias except hemolysis. Atypicalproerythroblasts predominate in megaloblastic anemia and erythremia.A right shift is seen in hemolytic conditions (nests of normoblasts, erythrons).Distribution and Cell Quality in Granulopoiesis. The same principle is validas for erythropoiesis: the more mature the cells, the greater proportion ofthe series they make up. A left shift indicates a greater than normal proportionof immature cells and a right shift a greater than normal proportionof mature cells (Table 4). Strong reactive conditions may lead to dissociationsin the maturation process, e.g., the nucleus shows the structure ofa myelocyte while the cytoplasm is still strongly basophilic. In malignancies,the picture is dominated by blasts, which may often be difficult toidentify with any certainty.A left shift is observed in all reactive processes and at the start of neoplastictransformation (smoldering anemia, refractory anemia withexcess blasts [RAEB]). In acute leukemias, undifferentiated and partiallymatured blasts may predominate. In agranulocytosis, promyelocytes aremost abundant.A right shift is diagnostically irrelevant.Cytochemistry. To distinguish between reactive processes and chronicmyeloid leukemia, leukocyte alkaline phosphatase is determined in freshsmears of blood. To distinguish between different types of acute leukemia,the peroxidase and esterase reactions are carried out (pp. 97 and 99), andiron staining is performed (p. 109) if myelodysplasia is suspected.Cytogenetic Analysis. This procedure will take the diagnosis forward incases of leukemia and some lymphadenomas. The fresh material must beheparinized before shipment, preferably after discussion with a specialistlaboratory.

The bone marrow contains a mixture of all the hematopoieticcellsabcFig. 18 Bone marrow cytology. a Bone marrow cytology of normal cell density ina young adult (smear from a bone marrow spicule shown at the lower right; magnification100). b More adipocytes with large vacuoles are present in this bonemarrow preparation with normal hematopoietic cell densities; usually found inolder patients. c Normal bone marrow cytology (magnification 400). Even thisoverview shows clearly that erythropoiesis (dense, black, round nuclei) accountsfor only about one-third of all the cells.55

56Normal Cells of the Blood and Hematopoietic OrgansQualitative and Quantitative Assessment of the Remaining Cells. Lymphocytecounts may be slightly raised in reactive processes, but a significantincrease suggests a disease of the lymphatic system. The exact classificationof these disease follows the criteria of lymphocyte morphology(Fig. 16). If elevated lymphocyte counts are found only in one preparationor within a circumscribed area, physiological lymph follicles in the bonemarrow are likely to be the source. In a borderline case, the histology andanalysis of lymphocyte surface markers yield more definitive data.Plasma cell counts are also slightly elevated in reactive processes andvery elevated in plasmacytoma. Reactive increase of lymphocytes andplasma cells with concomitant low counts in the other series is often anindication of panmyelopathy (aplastic anemia).Raised eosinophil and monocyte counts in bone marrow have the samediagnostic significance as in blood (p. 44).Megakaryocyte counts are reduced under the effects of all toxic stimulion bone marrow. Counts increase after bleeding, in essential thrombocytopenia(Werlhof syndrome), and in myeloproliferative diseases (chronicmyeloid leukemia, polycythemia, and essential thrombocythemia).Iron Staining of Erythropoietic Cells. Perls’ Prussian blue (also known asPerls’ acid ferrocyanide reaction) shows the presence of ferritin in 20–40%of all normoblasts, in the form of one to four small granules. The ironcontainingcells are called sideroblasts. Greater numbers of ferritingranules in normoblasts indicate a disorder of iron utilization (sideroachresia,especially in myelodysplasia), particularly when the granulesform a ring around the nucleus (ring sideroblasts). Perls’ Prussian blue reactionalso stains the diffuse iron precipitates in macrophages (Fig. 19 b).Under exogenous iron deficiency conditions the proportion of sideroblastsand iron-storing macrophages is reduced. However, if the shift iniron utilization is due to infectious and/or toxic conditions, the iron contentin normoblasts is reduced while the macrophages are loaded withiron to the point of saturation. In hemolytic conditions, the iron content ofnormoblasts is normal; it is elevated only in essential or symptomatic refractoryanemia (including megaloblastic anemia).

Not just the individual cell, but its relative proportion is relevantin bone marrow diagnosticsabcdFig. 19 Normal bone marrow findings. a Normal bone marrow: megakaryocyte(1), erythroblasts (2), and myelocyte (3). b Iron staining in the bone marrow cytology:iron-storing macrophage. c Normal bone marrow with slight preponderanceof granulocytopoiesis, e.g., promyelocyte (1), myelocyte (2), metamyelocyte (3),and band granulocyte (4). d Normal bone marrow with slight preponderance oferythropoiesis, e.g., basophilic erythroblast (1), polychromatic erythroblasts (2),and orthochromatic erythroblast (3). Compare (differential diagnosis) with theplasma cell (4) with its eccentric nucleus.57

58Normal Cells of the Blood and Hematopoietic OrgansBone Marrow: Medullary Stroma Cells➤ Fibroblastic reticular cells form a firm but elastic matrix in which theblood-forming cells reside, and are therefore rarely found in the bonemarrow aspirate or cytological smear. When present, they are mostlikely to appear as dense cell groups with long fiber-forming cytoplasmicprocesses and small nuclei. Iron staining shows them up as agroup of reticular cells which, like macrophages, have the potential tostore iron. If they become the prominent cell population in the bonemarrow, an aplastic or toxic medullary disorder must be considered.➤ Reticular histiocytes (not yet active in phagocytosis) are identical tophagocytic macrophages and are the main storage cells for tissueboundiron. Because of their small nuclei and easy-flowing cytoplasm,they are noticeable after panoptic staining only when they contain obviousentities such as lipids or pigments.➤ Osteoblasts are large cells with wide, eccentric nuclei. They differ fromplasma cells in that the cytoplasm has no perinuclear lighter space (cellcenter) and stains a cloudy grayish-blue. As they are normally rare inbone marrow, increased presence of osteoblasts in the marrow may indicatemetastasizing tumor cells (from another location).➤ Osteoclasts are multinucleated syncytia with wide layers of grayishbluestained cytoplasm, which often displays delicate azurophilicgranulation. They are normally extremely rare in aspirates, and whenthey are found it is usually under the same conditions as osteoblasts.They are distinguished from megakaryocytes by their round and regularnuclei and by their lack of thrombocyte buds.From the above, it will be seen that bone marrow findings can be assessedon the basis of a knowledge of the cells elements described above(p. 32 ff.), taking account of the eight categories given on p. 52 f. It alsoshows that a diagnosis from bone marrow aspirates can safely be madeonly in conjunction with clinical findings, blood chemistry, and the qualitativeand quantitative blood values. For this reason, a table of diagnosticsteps taking account of these categories is provided at the beginning ofeach of the following chapters.

Cells from the bone marrow stroma never occur in the bloodstreamabcFig. 20 Bone marrow stroma. a Spindle-shaped fibroblasts form the structuralframework of the bone marrow (shown here: aplastic hematopoiesis after therapyfor multiple myeloma). b A macrophage has phagocytosed residual nuclearmaterial (here after chemotherapy for acute leukemia). c Bone marrow osteoblastsare rarely found in the cytological assessment. The features that distinguishosteoblasts from plasma cells are their more loosely structured nuclei and thecloudy, “busy” basophilic cytoplasm. d Osteoclasts are multinucleated giant cellswith wide, spreading cytoplasm.59d

Abnormalities of the White Cell Series

62Abnormalities of the White Cell SeriesA very old-fashioned, intuitive way of classifying CBCs is to divide theminto those in which round to oval (mononuclear) cells predominate andthose in which segmented (polynuclear) cells predominate. However, thisold method does allow the relevant differential diagnosis to be inferredfrom a cursory screening of a slide.Differential Diagnostic NotesDifferential diagnosis when round to oval cells predominate➤ Reactive lymphocytoses and monocytoses, pp. 67 f., 89➤ Lymphatic system diseases (lymphomas, lymphocytic leukemia),p. 70➤ Acute leukemias, including episodes of chronic myeloid leukemia(CML), p. 96 ff.➤ Low counts of cells with segmented nuclei (agranulocytosis–aplastic anemia–myelodysplasia), pp. 86, 106, 148Differential diagnosis when segmented cells predominate➤ Reactive processes, p. 112 ff.➤ Chronic myeloid leukemia, p. 114 ff.➤ Osteomyelosclerosis, p. 122➤ Polycythemia vera, p. 162 ff.

Predominance of Mononuclear Round to Oval Cells63Predominance of Mononuclear Roundto Oval Cells (Table 5)The cell counts (per microliter) show a wide range of values and dependon a variety of conditions even in normal blood. Any disease may thereforeresult in higher (leukocytosis) or lower cell counts (leukopenia). Theassessment of cell counts requires the knowledge of normal values andtheir ranges (spreads) (Table 2, p. 12). The most important diagnostic indicatortherefore is not the cell count but the cell type. In a first assessment,mononuclear (round to oval) and polynuclear (segmented) cells arecompared. This is the first step in the differential diagnosis of the multifacetedspectrum of blood cells.Absolute or relative predominance of mononuclear cells points to a definedset of diagnostic probabilities. The differential diagnostic notes oppositecan help with a first orientation.Consequently, the most important step is to distinguish between lymphaticcells and myeloid blasts. The nuclear morphology makes it possibleto distinguish between the two cell types. In lymphatic cells the nucleususually displays dense coarse chromatin with slate-like architecture andlighter zones between very dense ones. In contrast, the immature cells inmyeloid leukemias contain nuclei with a more delicate reticular, sometimessand-like chromatin structure with a finer, more irregular pattern.

64Abnormalities of the White Cell SeriesTable 5 Diagnostic work-up for abnormalities in the white cell series with mononuclearcells predominatingClinical findings Hb MCH Leukocytes SegmentedcellsFever, lymph nodes,possibly exanthemaLymphocytes(%)n n ↓/n ↓ ↑StimulatedformsOther cells–Fever, severe lymphoma,possiblyspleen ↑Slowly progressinglymph node enlargementin several locationsn n ↑ ↓ ↑ Large blasticstimulated cells(DD: lymphoblasts)↓/n ↓/n ↑ ↓ ↑↑ –Night sweat, slowlyprogressing lymphoma(spleen),possibly fever↓ ↓ n/↑ ↓ ↑ Plasmacytoidcells, possiblyrouleaux formationof theerythrocytesSlowly progressinglymph node enlargementin one or a fewlocationsIsolated severesplenomegaly↓ n/↓ n/↑/↓ n/↓ Possibly ↑ Possibly cellswith grooves↓ ↓ ↓/↑ ↓ Hairy cellsFever, angina n n ↓ ↓ ↑ Monocytes ↑Diffuse generalsymptoms↓ ↓ ↑ ↓ n Monocytes ↑Pale skin, fever(signs of hemorrhage)↓ ↓ ↑/n/↓ ↓ ↓ Atypical roundcells predominateFatigue, nightsweat, possiblybone pain↓ ↓ n/↓ n n Possibly rouleauxformations oferythrocytesDiagnostic steps proceed from left to right. | The next step is usually unnecessary; optionalstep, may not progress the diagnosis; ⎯→ the next step is obligatory.

Predominance of Mononuclear Round to Oval Cells65ThrombocytesElectrophoresisTentativediagnosisn n/Y↑ Virus infection,e.g., rubeolan Y↑ MononucleosisEvidence/advanceddiagnosticsClinical developments,possibly serologicaltestsMononucleosis quicktest, EBV serology(cytomegaly titer?)Bone marrowRef. pagep. 66p. 68n/↓ n/Y↓ Leukemicnon-Hodgkinlymphoma,esp. CLLn/↓PossiblyY↑n/↓ n Non-Hodgkinlymphoma,e.g. follicularlymphoma↓ n Hairy cellleukemian n AgranulocytosisnPossiblya 2↑Reactivemonocytosisin chronicinfection ortumor↓ n Acuteleukemian/↓Sharppeaks ↑MultiplemyelomaMarker analysis ofperipheral lymphocytesis sufficient in typicaldisease presentations;lymph node histologyfor treatment decisions,if necessaryAlwaysinvolved inCLL, notalways inother lymphomasImmunocytoma(incl.Waldenströmdisease)Immunoelectrophoresis;marker analysis forlymphocytes in bloodand bone marrow;lymph node histology incase of doubtLymph node histologyCell surface markers⎯⎯⎯⎯⎯⎯⎯⎯→Determine diseaseoriginCytochemistry, markeranalysis, possibly cytogenetics⎯⎯⎯⎯⎯⎯⎯⎯→Immunoelectrophoresis,skeletal X-ray⎯⎯⎯⎯⎯⎯⎯⎯→.Usuallyinvolved.For the purposeof staging,possiblypositive.Alwaysinvolved, discretein thebeginning.MaturationarrestMarker forblastsBone marrowcontainsplasma cellsp. 74p. 78p. 78p. 80p. 86p. 88p. 90ffp. 82

66Abnormalities of the White Cell SeriesReactive LymphocytosisLymphatic cells show wide variability and transform easily. This is usuallyseen as enlarged nuclei, a moderately loose, coarse chromatin structure,and a marked widening of the basophilic cytoplasmic layer.Clinical findings, which include acute fever symptoms, enlarged lymphnodes, and sometimes exanthema, help to identify a lymphatic reactivestate. Unlike the case in acute leukemias, erythrocyte and thrombocytecounts are not significantly reduced. Although the granulocyte count isrelatively reduced, its absolute value (per microliter) rarely falls below thelower limit of normal values.Morphologically normal lymphocytes predominate in the blood analyses inthe following diseases:➤ Whooping cough (pertussis) with clear leukocytosis and total lymphocytecounts up to 20 000 and even 50 000/µl; occasionally, slightlyplasmacytoid differentiation.➤ Infectious lymphocytosis, a pediatric infectious disease with fever ofshort duration. Lymphocyte counts may increase to 50 000/µl.➤ Chickenpox, measles, and brucellosis, in which a less well-developedrelative lymphocytosis is found, and the counts remain within the normalrange.➤ Hyperthyroidism and Addison disease, which show relative lymphocytosis.➤ Constitutional relative lymphocytosis, which can reach up to 60% andoccurs without apparent reason (mostly in asthenic teenagers).➤ Absolute granulocytopenias with relative lymphocytosis (p. 86).➤ Chronic lymphocytic leukemia (CLL), which is always accompanied byabsolute and relative lymphocytosis, usually with high cell counts.Transformed, “stimulated” lymphocytes (“virocytes”) predominate in theCBC in the following diseases with reactive symptoms:➤ Lymphomatous toxoplasmosis does not usually involve significantleukocytosis. Slightly plasmacytoid cell forms are found.➤ In rubella infections the total leukocyte count is normal or low, and thelymphocytosis is only relatively developed. The cell morphology rangesfrom basophilic plasmacytoid cells to typical plasma cells.➤ In hepatitis the total leukocyte and lymphocyte counts are normal.However, the lymphocytes often clearly show plasmacytoid transformation.➤ The most extreme lymphocyte transformation is observed in mononucleosis(Epstein–Barr virus [EBV] or cytomegalovirus [CMV] infection)(p. 68).

During lymphatic reactive states, variable cells with dense,round nuclei (e.g., virocytes) dominate the CBCabcdeFig. 21 Lymphatic reactive states. a–e Wide variability of the lymphatic cells in alymphotropic infection (in this case cytomegalovirus infection). Some of the cellsmay resemble myelocytes, but their chromatin is always denser than myelocytechromatin.67

68Abnormalities of the White Cell SeriesExamples of Extreme Lymphocytic Stimulation:Infectious MononucleosisEpstein–Barr virus infection should be considered when, after a prodromalfever of unknown origin, there are signs of enlarged lymph nodes anddeveloping angina, and the blood analysis shows predominantly mononuclearcells and a slightly, or moderately, elevated leukocyte count. Varyingproportions of the mononuclear cells (at least 20%) may be rather extensivelytransformed round cells (Pfeiffer cells, virocytes). Immunologicalmarkers are necessary to ascertain that these are stimulated lymphocytes(mostly T-lymphocytes) defending the B-lymphocyte stem populationagainst the virus attack. The nuclei of these stimulated lymphocytes aretwo- to three-fold larger than those of normal lymphocytes and theirchromatin has changed from a dense and coarse structure to a looser,more irregular organization. The cytoplasm is always relatively wide andmore or less basophilic with vacuoles. Granules are absent. A small proportionof cells appear plasmacytoid. In the course of the disease, thedegree of transformation and the proportions of the different cell morphologieschange almost daily. A slight left shift and elevated monocytecount are often found in the granulocyte series.Acute leukemia is often considered in the differential diagnosis in additionto other viral conditions, because the transformed lymphocytes canresemble the blasts found in leukemia. Absence of a quantitative reductionof hematopoiesis in all the blood cell series, however, makes leukemiaunlikely, as do the variety and speed of change in the cell morphology. Finally,serological tests (EBV antigen test, test for antibodies, and, if indicated,quick tests) can add clarification.Where serological tests are negative, the cause of the symptoms is usuallycytomegalovirus rather than EBV.Characteristics of Infectious MononucleosisAge of onset: School ageClinical findings: Enlarged lymph nodes (rapid onset), inflammationsof throat and possibly spleen CBC: Leukocytes , stimulated lymph nodes, partially lymphoblasts(hematocrit and thrombocytes are normal)Further diagnostics: EBV serological test (IgM +), transaminases usuallyDifferential diagnosis: Lymphomas (usually without fever), leukemia(usually Hb , thrombocytes ) Persistent disease (more than 3weeks): possibly test for blood cell markers, lymph node cytology( histology)Course, treatment: Spontaneous recovery within 2–4 weeks; generalpalliation

Extreme transformation of lymphocytes leads to blast-like cells:mononucleosisabcdFig. 22 Lymphocytes during viral infection. a “Blastic,” lymphatic reactive form(Pfeiffer cell), in addition to less reactive virocytes in Epstein–Barr virus (EBV) infection.This phase with blastic cells lasts only a few days. b Virocyte (1) with homogeneousdeep blue stained cytoplasm in EBV infection, in addition to normallymphocyte (2) and monocyte (3). c Virus infection can also lead to elevatedcounts of large granulated lymphocytes (LGL) (1). Monocyte (2). d Severelymphatic stress reaction with granulated lymphocytes. A lymphoma must beconsidered if this finding persists.69

70Abnormalities of the White Cell SeriesDiseases of the Lymphatic System(Non-Hodgkin Lymphomas)Malignant diseases of the lymphatic system are further classified as Hodgkinand non-Hodgkin lymphomas (NHL). NHL will be discussed here becausemost NHLs can be diagnosed on the blood analysis.➤ Non-Hodgkin lymphomas arise mostly from small or blastic B-cells.➤ Small-cell NHL cells are usually leukemic and relatively indolent, of thetype of chronic lymphocytic leukemia and its variants.➤ Blastic NHL (precursor lymphoma) is not usually leukemic. An exceptionis the lymphoblastic lymphoma, which takes its course as an acutelymphocytic leukemia.➤ Plasmacytoma is an osteotropic B-cell lymphoma that releases not itscells but their products (immunoglobulins) into the bloodstream.➤ Malignant lymphogranulomatosis (Hodgkin disease) cannot be diagnosedon the basis of blood analysis. It is therefore discussed underlymph node cytology (p. 176).The modern pathological classification of lymphomas is based on morphologyand cell immunology. The updated classification system accordingto Lennert (Kiel) has been adopted in Germany and was recently modifiedto reflect the WHO classification. The definitions of stages I–IV in NHLagree with the Ann Arbor classification for Hodgkin’s disease.Classification of non-Hodgkin: comparison of the relevant classes in the Kiel and WHO classifica-Table 6ationsWHO Kiel ClinicalCharacteristicsMarker*Precursor lymphomas/leukemias➤➤Precursor-B-lymphoblasticlymphomaPrecursor B-cell active lymphoblasticleukemiaMature B-cell lymphoma➤Chronic lymphocytic leukemia– contains the lymphoplasmacytoidimmunocytomaB-lymphoblasticlymphomaB-ALLB-CLLLymphoplasmacytoidimmunocytomaAggressiveUsually indolentUsually indolent➤ B-cell prolymphocytic leukemia AggressiveTdt,CD19, 22,79 aCD5, 19,20, 23tris 12 or13 q-,11 q-, 6 q-,p 53

Predominance of Mononuclear Round to Oval Cells71Table 6aContinuedWHO Kiel ClinicalCharacteristicsMarker*➤ Lymphoplasmacytic leukemia LymphoplasmacyticimmunocytomaSometimes IgMparaprotein(Waldenströmdisease)➤ Mantle cell lymphoma Centrocytic lymphoma Usually aggressive➤➤Marginal zone lymphoma– Nodal– Extranodal in mucosa (MALT)– Lienal (splenic)Follicular lymphoma– Grade 1, 2– Grade 3 (a, b)Monocytoid lymphomaMALT (mucosa-assoc.lymphoid tissue) lymphomaLymphoma withsplenomegalyCentroblastic/centrocyticlymphoma;centroblastic lymphoma(a), secondary (b), follicularUsually aggressiveOften indolentUsually indolentHighly malignant–/+ CD5– CD23,CD5t(11;14)bcl-1rearr.–CD5,CD23–CD5, CD10t(14;18)bcl2➤ Hairy cell leukemia Hairy cell leukemia Usually indolent –CD103,CD11 c,CD25t(2;8)t(8;14)➤Plasma cell myeloma (plasmacytoma)– Monoclonal hypergammaglobulinemia(GUS)– Solitary bone plasmacytoma– Extraosseous bone plasmacytoma– Primary amyloidosis– Heavy-chain disease[Plasmacytoma is notincluded in the Kielclassification]CD 138Primary large-cell lymphoma➤Diffuse large-cell B-celllymphoma– Centroblastic– Immunoblastic– Large-cell anaplasticCentroblasticImmunoblasticLarge-cell anaplasticExtremely malignantExtremely malignantExtremely malignant➤ Burkitt lymphoma Burkitt lymphoma Extremely malignantCD20,79 a, 19,22t(2;8)t(8;14)myc* Cited markers are positive, absent markers are indicated with a minus sign.

72Abnormalities of the White Cell SeriesTable 6b T-cell lymphomas (since T-cell lymphomas make up only 10% of all NHLs, this table gives just a briefcharacterization; for markers see Table 7)WHO( Kiel)Classification➤ T-prolymphocytic leukemia➤ Large granular lymphocyte leukemia (LGL)➤ T-cell lymphoblastic leukemiaManifestationLeukemicLeukemicLeukemicClinical characteristicsAggressiveSometimes indolentAggressive➤ Sézary syndrome, mycosis fungoides Cutaneous Chronic, progressive➤ Angioimmunoblastic T-cell lymphoma (AILD) Nodal and ENT Usually aggressive➤ Lymphoblastic T-cell lymphoma➤ T-cell zone lymphoma (nonspecific peripherallymphoma)➤ Lennert lymphoma with multifocal epithelioidcells➤ Large-cell anaplastic lymphoma (ki1)NodalNodalNodalNodalAggressiveSometimes slowlyprogressiveSometimes slowlyprogressiveAggressiveDifferentiation of the Lymphatic Cells and Cell SurfaceMarker Expression in Non-Hodgkin Lymphoma CellsNon-Hodgkin lymphoma cells derive monoclonally from specific stages inthe B- or T-cell differentiation, and their surface markers reflect this. Thesurface markers are identified in immunocytological tests (Table 7) carriedout on heparinized blood or bone marrow spicules.The blastic lymphomas will not be discussed further in the context ofdiagnostics based on blood cell morphology. The findings in the primarilyleukemic forms of the disease, such as lymphoblastic lymphoma, resemblethose for ALL (p. 104). Other blastic lymphomas can usually onlybe diagnosed on the basis of lymph node tissue (Fig. 65). Of course, despiteall the progress in the analysis of blood cell differentiation, often analysisof histological slides in conjunction with the blood analysis is required fora confident diagnosis.

Predominance of Mononuclear Round to Oval Cells73Table 7 Cell surface markers of lymphatic cells in leukemic, low-grade malignant non-Hodgkin lymphomaMarker B-CLL B-PLL HCL FL MCL SLVL T-CLL GL SS T-PLL ATLLS Ig (+) ++ ++ ++ ++ ++ – – – – –CD 2 – – – – – – + + + + +CD 3 – – – – – – + – + + +CD 4 – – – – – – – – + +/– +CD 5 ++ – – – + – – – + + +CD 7 – – – – – – – – +/– + –/+CD 8 – – – – – – + +/– +/– +/– +/–CD 19/20/24 ++ ++ ++ + + ++ – – – – –CD 22 +/– ++ ++ + + ++ – – – – –CD 10 – – – +/– – – – – – – –CD 25 – – ++ – – +/– – – – – +CD 56 – – – – – – – + – – –CD 103 – – ++ – – – – – – – –CLL chronic lymphocytic leukemia; PLL prolymphocytic leukemia; HCL hairy cell leukemia; FL follicular lymphoma; MCL mantle cell lymphoma; SLVL splenic lymphoma with villous lymphocytes;LGL large granular lymphocyte leukemia; SS Sézary syndrome; ATLL adult T-cell lymphoma.

74Abnormalities of the White Cell SeriesChronic Lymphocytic Leukemia (CLL) and Related DiseasesA chronic lymphadenoma, or chronic lymphocytic leukemia, can sometimesbe clinically diagnosed with some certainty. An example is the caseof a patient (usually older) with clearly enlarged lymph nodes and significantlymphocytosis (in 60% of the cases this is greater than 20 000/µl andin 20% of the cases it is greater than 100 000/µl) in the absence of symptomsthat point to a reactive disorder. The lymphoma cells are relativelysmall, and the nuclear chromatin is coarse and dense. The narrow layer ofslightly basophilic cytoplasm does not contain granules. Shadows aroundthe nucleus are an artifact produced by chromatin fragmentation duringpreparation (Gumprecht’s nuclear shadow). In order to confirm the diagnosis,the B-cell markers on circulating lymphocytes should be characterizedto show that the cells are indeed monoclonal. The transformedlymphocytes are dispersed at varying cell densities throughout the bonemarrow and the lymph nodes. A slowly progressing hypogammaglobulinemiais another important indicator of a B-cell maturation disorder.Transition to a diffuse large-cell B-lymphoma (Richter syndrome) israre: B-prolymphocytic leukemia (B-PLL) displays unique symptoms. Atleast 55% of the lymphocytes in circulating blood have large centralvacuoles. When 15–55% of the cells are prolymphocytes, the diagnosis ofatypical CLL, or transitional CLL/PLL is confirmed. In some CLL-like diseases,the layer of cytoplasm is slightly wider. B-CLL was defined as lymphoplasmacytoidimmunocytoma in the Kiel classification. According tothe WHO classification, it is a B-CLL variation (compare this with lymphoplasmacyticleukemia, p. 78). CLL of the T-lymphocytes is rare. The cellsshow nuclei with either invaginations or well-defined nucleoli (T-prolymphocyticleukemia). The leukemic phase of cutaneous T-cell lymphoma(CTCL) is known as Sézary syndrome. The cell elements in this syndromeand T-PLL are similar.Fig. 23 CLL. a Extensive proliferation of lymphocytes with densely structured nuclei and little variation in CLL. Nuclear shadows are frequently seen, a sign ofthe fragility of the cells (magnification 400). b Lymphocytes in CLL with typicalcoarse chromatin structure and small cytoplasmic layer (enlargement of a sectionfrom 23 a, magnification 1000); only discreet nucleoli may occur. c Slightlyeccentric enlargement of the cytoplasm in the lymphoplasmacytoid variant ofCLL.

Monotonous proliferation of small lymphocytes suggests chroniclymphocytic leukemia (CLL)abcdeFig. 23 d Proliferation of atypical large lymphocytes (1) with irregularly structurednucleus, well-defined nucleolus, and wide cytoplasm (atypical CLL or transitionalform CLL/PLL). e Bone marrow cytology in CLL: There is always strongproliferation of the typical small lymphocytes, which are usually spread out diffusely.75

76Abnormalities of the White Cell SeriesTable 8 Staging of CLL according to Rai (1975)StageIdentifying criteria/definition(Low risk) 0 Lymphocytosis 15 000/µlBone marrow infiltration 40%(Intermediaterisk)IIILymphocytosis and lymphadenopathyLymphocytosis and hepatomegaly and/or splenomegaly(with or without lymphadenopathy)(High risk) III Lymphocytosis and anemia (Hb 11.0 g/dl) (with orwithout lymphadenopathy and/or organomegaly)IV Lymphocytosis and thrombopenia ( 100 000/µl)(with or without anemia, lymphadenopathy, ororganomegaly)Table 9 Staging of CLL according to Binet (1981)StageABCIdentifying criteria/definitionHb 10.0 g/dl, normal thrombocyte count 3 regions with enlarged lymph nodesHb 10.0 g/dl, normal thrombocyte count 3 regions with enlarged lymph nodesHb 10.0 g/dl and/or thrombocyte count 100 000/µlindependent of the number of affected locationsCharacteristics of CLLAge of onset: Mature adulthoodClinical presentation: Gradual enlargement of all lymph nodes, usuallymoderately enlarged spleen, slow onset of anemia and increasingsusceptibility to infections, later thrombocytopeniaCBC: In all cases absolute lymphocytosis; in the course of the diseaseHb , thrombocytes , immunoglobulin Further diagnostics: Lymphocyte surface markers (see pp. 68 ff.); bonemarrow (always infiltrated); lymph node histology further clarifiesthe diagnosisDifferential diagnosis: (a) Related lymphomas: marker analysis,lymph node histology; (b) acute leukemia: cell surface marker analysis,cytochemistry, cytogenetics (pp. 88 ff.)Course, therapy: Individually varying, usually fairly indolent course;in advanced stages or fast progressing disease: moderate chemotherapy(cell surface marker, see Table 7)

Atypical lymphocytes are not part of B-CLLabcdeFig. 24 Lymphoma of the B-cell and T-cell lineages. a Prevalence of large lymphocyteswithclearlydefinednucleoliandwidecytoplasm:prolymphocyticleukemiaofthe B-cell series (B-PLL). b The presence of large blastic cells (arrow) in CLL suggest arare transformation (Richter syndrome). c The rare Sézary syndrome (T-cell lymphomaof the skin) is characterized by irregular, indented lymphocytes. d Prolymphocyticleukemia of the T-cell series (T-PLL) with indented nuclei and nucleoli(rare). e Bone marrow in lymphoplasmacytic immunocytoma: focal or diffuse lymphocyteinfiltration (e.g., 1), plasmacytoid lymphocytes (e.g., 2) and plasma cells(e.g., 3). Red cell precursors predominate (e.g., basophilic erythroblasts, arrow).77

78Abnormalities of the White Cell SeriesThe pathological staging for CLL is always Ann Arbor stage IV because thebone marrow is affected. In the classifications of disease activity by Raiand Binet (analogous to that for leukemic immunocytoma), the transitionbetween stages is smooth (Tables 8 and 9).Lymphoplasmacytic LymphomaThe CBC shows lymphocytes with relatively wide layers of cytoplasm. Thebone marrow contains a mixture of lymphocytes, plasmacytic lymphocytes,and plasma cells. In up to 30% of cases paraprotein is secreted, predominantlymonoclonal IgM. This constitutes the classic Waldenströmsyndrome (Waldenström macroglobulinemia). The differential diagnosismay call for exclusion of the rare plasma cell leukemia (see p. 82) and oflymphoplasmacytoid immunocytoma, which is closely related to CLL (seep. 74).Characteristics➤ Lymphoplasmacytoid immunocytoma: This is a special form of B-CLL in which usually only a few precursors migrate into the bloodstream(a lesser degree of malignancy). A diagnosis may only bepossible on the basis of bone marrow or lymph node analysis.➤ Lymphoplasmacytic lymphoma: Few precursors migrate into thebloodstream (i.e., bone marrow or lymph node analysis is sometimesnecessary). There is often secretion of IgM paraprotein,which can lead to hyperviscosity.Further diagnostics: Marker analyses in circulating cells, lymph node cytology,bone marrow cytology and histology, and immunoelectrophoresis.Plasmacytoma cells migrate into the circulating blood in appreciablenumbers in only 1–2% of all cases of plasma cell leukemia. Therefore, paraproteinsmust be analyzed in bone marrow aspirates (p. 82).Facultative Leukemic Lymphomas(e.g., Mantle Cell Lymphoma and Follicular Lymphoma)In all cases of non-Hodgkin lymphoma, the transformed cells may migrateinto the blood stream. This is usually observed in mantle cell lymphoma:The cells are typically of medium size. On close examination, their nucleishow loosely structured chromatin and they are lobed with small indentations(cleaved cells). Either initially, or, more commonly, during the courseof the disease, a portion of cells becomes larger with relatively enlargednuclei (diameter 8–12 µm). These larger cells are variably “blastoid.” Lymphoidcells also migrate into the blood in stage IV follicular lymphoma.

Deep nuclear indentation suggests follicular lymphoma ormantle cell lymphomaabcFig. 25 Mantle cell lymphoma. a Fine, dense chromatin and small indentationsof the nuclei suggest migration of leukemic mantle cell lymphoma cells into theblood stream. b Denser chromatin and sharp indentations suggest migration offollicular lymphoma cells into the blood stream. c Diffuse infiltration of the bonemarrow with polygonal, in some cases indented lymphatic cells in mantle celllymphoma. Bone marrow involvement in follicular lymphoma can often only bedemonstrated by histological and cytogenetic studies.79

80Abnormalities of the White Cell Series“Monocytoid” cells with a wide layer of only faintly staining cytoplasmoccur in blood in marginal zone lymphadenoma (differential diagnosis:lymphoplasmacytic immunocytoma).Lymphoma, Usually with Splenomegaly(e.g., Hairy Cell Leukemia and Splenic Lymphomawith Villous Lymphocytes)Hairy cell leukemia (HCL). In cases of slowly progressive general malaisewith isolated splenomegaly and pancytopenia revealed by CBC (leukocytopenia,anemia, and thrombocytopenia), the predominating mononuclearcells deserve particular attention. The nucleus is oval, often kidney beanshaped,and shows a delicate, elaborate chromatin structure. The cytoplasmis basophilic and stains slightly gray. Long, very thin cytoplasmicprocesses give the cells the hairy appearance that gave rise to the term“hairy cell leukemia” used in the international literature. The disease affectsthe spleen, liver, and bone marrow. Severe lymphoma is usually absent.Aside from the typical hairy cells with their long, thin processes,there are also cells with a smooth plasma membrane, similar to cells in immunocytoma.A variant shows well-defined nucleoli (HCL-V, hairy cellleukemia variant). A bone marrow aspirate often does not yield materialfor an analysis (“punctio sicca” or “empty tap”) because the marrow is veryfibrous. Apart from the bone marrow histology, advanced cell diagnosticsare therefore very important, in particular in the determination of bloodcell surface markers (immunophenotyping). This analysis reveals CD 103and 11 c as specific markers and has largely replaced the test for tartrateresistantacid phosphatase.Splenic lymphoma with villous lymphocytes (SLVL). This lymphatic systemdisease mostly affects the spleen. There is little involvement of the bonemarrow and no involvement of the lymph nodes. The blood contains lymphaticcells, which resemble hairy cells. However, the “hairs,” i.e., cytoplasmicprocesses, are thicker and mostly restricted to one area at the cellpole, and the CD 103 marker is absent.Splenomegaly may develop in all non-Hodgkin lymphomas. In hairy cellleukemia, the rare splenic lymphadenoma with villous lymphocytes(SLVL) and marginal zone lymphadenoma may be seen. These mostly affectthe spleen.

Cytoplasmic processes the main feature of hairy cell leukemiaabdceFig. 26 Hairy cell leukemia and splenic lymphoma. a and b Ovaloid nuclei andfinely “fraying” cytoplasm are characteristics of cells in hairy cell leukemia (HCL).c Occasionally, the hairy cell processes appear merely fuzzy. d and e When the cytoplasmicprocesses look thicker and much less like hair, diagnosis of the rare spleniclymphoma with villous lymphocytes (SLVL) must be considered. Here, too, thenext diagnostic step is analysis of cell surface markers.81

82Abnormalities of the White Cell SeriesMonoclonal Gammopathy (Hypergammaglobulinemia),Multiple Myeloma*, Plasma Cell Myeloma, PlasmacytomaPlasmacytoma is the result of malignant transformation of the mostmature B-lymphocytes (Fig. 1, p. 2). For this reason the diagnostics of thisdisease will be discussed here, even though migration of its specific cellsinto the blood stream (plasma cell leukemia) is extremely rare (1–2%).Immunoelectrophoresis of serum and urine is performed when electrophoresisshows very discrete gammaglobulin, or globulin, fractions, orwhen hypogammaglobulinemia is found (in light-chain plasmacytoma). Awide range of possibilities arises for the differential diagnosis of monoclonaltransformed cells (Table 10).The presence of more than 10% of plasma cells, or atypical plasma cellsin the bone marrow, is an important diagnostic factor in the diagnosis ofplasmacytoma. For more criteria, see p. 84.Table 10Differential diagnosis of monoclonal hypergammaglobulinemiaTypeBenign disorders➤ Essential hypergammaglobulinemia= MGUS (monoclonal gammopathyof unknown significance)➤ Symptomatic hypergammaglobulinemia,secondary to– Infections– Tumors– Autoimmune diseaseMalignant diseases➤ Plasmocytoma(usually IgG, A or light-chain[Bence Jones protein], rarely IgM,D, E) 90% disseminated (multiplemyeloma), 5% solitary, 5%extramedullary (like a lymphomaor ENT tumor)➤ Lymphomae. g., immunocytoma, CLL(potentially all lymphomas of theB-cell series)CharacteristicsUsually in advanced age 10%, plasma cells found in thebone marrow, no progression,normal polyclonal IgAll ages (otherwise as above)– Osteolysis or X-ray with severeosteoporosis– Plasmocytosis of the bone marrow 10%– Monoclonal gammaglobulin inserum/urine with progression– Enlarged lymph nodes– Usually blood lymphocytosis– Monoclonal immunoglobulin,usually IgM* The current WHO classification suggests “multiple myeloma” (MM) for generalizedplasmacytoma and “plasmacytoma” only for the rare solitary or nonosseous form ofplasmacytoma.

Plasmacytoma cannot be diagnosed without bone marrow analysisabFig. 27 Reactive plasmacytosis and plasmacytoma. a Bone marrow cytologywith clear reactive features in the granulocyte series: strong granulation of promyelocytes(1) and myelocytes (2), eosinophilia (3), and plasma cell proliferation(4): reactive plasmacytosis (magnification 630). b Extensive (about 50%) infiltrationof the bone marrow of mostly well-differentiated plasma cells: multiplemyeloma (magnification 400).83

84Abnormalities of the White Cell SeriesVariability of Plasmacytoma MorphologyIt is not easy to visually distinguish malignant cells from normal plasmacells. Like lymphocytes, normal plasma cells have a densely structured nucleus.Plasma cell nuclei with radial chromatin organization, known as“wheel-spoke nuclei,” are mostly seen during histological analysis.The following attributes suggest a malignant character of plasma cells:the cells are unusually large (Fig. 28 c), they contain crystalline inclusionsor protein inclusions (“Russell bodies”) (Fig. 28 b), or they have more thanone nucleus (Fig. 28 c).In the differential diagnosis, they must be distinguished from hematopoieticprecursor cells (Fig. 28 d), osteoblasts (Fig. 20 c), and blasts in acuteleukemias (see Fig. 31, p. 97).Bone marrow involvement may be focal or in rare cases even solitary.Aside from cytological tests, bone marrow histology assays are thereforeindicated. Sometimes, the biopsy must be obtained from a clearly identifiedosteolytic region.Although plasmacytomas progress slowly, staging criteria are available(staging according to Salmon and Durie) (Table 11).Therapy may be put on hold in stage I. Smoldering indolent myelomacan be left for a considerable time without the introduction of therapystress. However, chemotherapy is indicated once the myeloma hasexceeded the stage 1 criteria.Table 11Staging of plasmacytomas according to Salmon and DurieStage IAll the following are present:– Hb 10 g/dl– Serum calcium is normal– X-ray shows normal bone structureor solitary skeletal plasmacytoma– IgG 5 g/dl*IgA 3 g/dl*Light chains in the urine* 4 g/24 h* Monoclonal in each case.Stage IIFindings fulfill neither stage I norstage III criteriaStage IIIOne or more of the following arepresent:– Hb 8.5 g/dl– Serum calcium is elevated– X-ray shows advanced bone lesions– IgG 7 g/dl*IgA 5 g/dl*Light chains in the urine: 12 g/24 h

Atypias and differential diagnoses of multiple myelomaabcdFig. 28 Atypical cells in multiple myeloma. a Extensive infiltration of the bonemarrow by loosely structured, slightly dedifferentiated plasma cells with wide cytoplasmin multiple myeloma. b In multiple myeloma, vacuolated cytoplasmicprotein precipitates (Russell bodies) may be seen in plasma cells but are withoutdiagnostic significance. c Binuclear plasma cells are frequently observed in multiplemyeloma (1). Mitotic red cell precursor (2). d Differential diagnosis: red cellprecursor cells can sometimes look like plasma cells. Proerythroblast (1) and basophilicerythroblast (2).85

86 Abnormalities of the White Cell SeriesRelative Lymphocytosis Associated withGranulocytopenia (Neutropenia) and AgranulocytosisNeutropenia is defined by a decrease in the number of neutrophilicgranulocytes with segmented nuclei to less than 1500/µl (1.5 10 9 /l). Aneutrophil count of less than 500/µl (0.5 10 9 /l) constitutes agranulocytosis.Absolute granulocytopenias with benign cause develop into relativelymphocytoses.In the most common clinical picture, drug-induced acute agranulocytosis,the bone marrow is either poor in cells or lacks granulopoietic precursorcells (aplastic state), or shows a “maturation block” at the myeloblast–promyelocytestage. The differential diagnosis of pure agranulocytosisversus aplasias of several cell lines is outlined on page 146.Classification of Neutropenias and Agranulocytoses1. Drug-induced:a) Dose-independent—acute agranulocytosis. Caused by hypersensitivityreactions: for example to pyrazolone, antirheumatic drugs(anti-inflammatory agents), antibiotics, or thyrostatic drugs.b) Relatively dose-dependent—subacute agranulocytosis (observed forcarbamazepine [Tegretol], e.g. antidepressants, and cytostaticdrugs).c) Dose-dependent—cytostatic drugs, immunosuppressants.2. Infection-induced:a) E.g., EBV, hepatitis, typhus, brucellosis.3. Autoimmune neutropenia:a) With antibody determination of T-cell or NK-cell autoimmune responseb) In cases of systemic lupus erythematosus (SLE), Pneumocystis cariniipneumonia (PCP), Felty syndromec) In cases of selective hypoplasia of the granulocytopoiesis (“purewhite cell aplasia”)4. Congenital and familial neutropenias:Various pediatric forms; sometimes not expressed until adulthood,e.g. cyclical neutropenia5. Secondary neutropenia in bone marrow disease:Myelodysplastic syndromes (MDS), e.g., acute leukemia, plasmacytoma,pernicious anemia

Bone marrow diagnosis is indicated in cases of unexplained agranulocytosisabFig. 29 The bone marrow in agranulocytosis. a In the early phase of agranulocytosisthe bone marrow shows only red cell precursor cells (e.g., 1), plasma cells(2), and lymphocytes (3); in this sample a myeloblast—a sign of regeneration—isalready present (4). b Bone marrow in agranulocytosis during the promyelocyticphase, showing almost exclusively promyelocytes (e.g., 1); increased eosinophilicgranulocytes (2) are also present.87

88Abnormalities of the White Cell SeriesMonocytosisIf mononuclear cells stand out in showing an unusually elaborate nuclearstructure with ridges and lobes and a wider cytoplasmic layer with verydelicate granules (for characteristics see p. 46, for cell function, see p. 6),and this is in the context of relative ( 10%) or absolute monocytosis (cellcount 900/µl), a series of possible triggers must be considered (Table12). If the morphology does not clearly identify the cells as monocytes,then esterase assays should be done in a hematological laboratory usingunstained smears.Table 12Possible causes of monocytosisInfectionNonspecific monocytosis occurs inmany bacterial infections duringrecuperation from or in the chronicphase of:– Mononucleosis (aside from stimulatedlymphocytes there are alsomonocytes)– Listeriosis– Acute viral hepatitis– Parotitis epidemica (mumps)– Chickenpox– Recurrent fever– Syphilis– Tuberculosis– Endocarditis lenta– Brucellosis (Bang disease)– Variola vera (smallpox)– Rocky mountain spotted fever– Malaria– Paratyphoid fever– Kala-azar– Thypus fever– Trypanosomiasis (African sleepingsickness)Chronic reactive condition of theimmune system, e. g., in:– Autoimmune diseases– Chronic dermatoses– Regional ileitis– SarcoidosisParaneoplasm(as attempted immune defense),e. g., in:– Large solid tumors– LymphogranulomatosisNeoplasm– Chronic myelomonocyticleukemia (CMML, p. 107)– Acute monocytic leukemia(p. 100)– Acute myelomonocytic leukemia(p. 98)

Conspicuously large numbers of monocytes usually indicate areactive disorder. Only a monotonous predominance of monocytessuggests leukemiaabcFig. 30 Reactive monocytosis and monocytic leukemia. a Reactive and neoplasticmonocytes are morphologically indistinguishable; here two relatively condensedmonocytes in reactive monocytosis are shown. b Whenever monocytes arefound exclusively, a malignant etiology is likely: in this case AML M 5 b according tothe FAB classification (see p. 100). Auer bodies (arrow). c Monocytes of differentdegrees of maturity, segmented neutrophilic granulocytes (1), and a small myeloblast(2) in chronic myelomonocytic leukemia (CMML, see p. 107).89

90Abnormalities of the White Cell SeriesAcute LeukemiasAcute leukemias are described in this place for morphological reasons, becausethey involve a predominance of mononuclear cells (p. 63). Although—orperhaps because—the term “leukemia” is relatively imprecise,an overview seems required (Table 13).The cellular phenomenon common to the different forms of leukemia isthe rapidly progressive reduction in numbers of mature granulocytes,thrombocytes, and erythrocytes. Simultaneously, the leukocyte countusually increases due to the occurrence of atypical round cells.Table 13Overview of all forms of leukemiaType of leukemiaClassification/clinical findingsAcute leukemias (ALAML, ALL)– Myeloid M 0-7 (incl. monocytic, erythroid,megakaryoblastic leukemias)Always acute disease, often withfever and tendency to hemorrhage– Lymphocytic There may be lymphoma and thymusinfiltratesChronic myeloid leukemia (CML)– Persistent leukemic diseases of themyeloproliferative system (p. 114)Chronic lymphocytic leukemia (CLL)and other leukemic lymphomas– B-CLL (90%), T-CLL (p.74 f.)– Prolymphocytic leukemia (p.77)– Hairy cell leukemia (p.80)Chronic disease, usually withsplenomegaly– Chronic lymphocytic leukemia,(rarely) prolymphocyticleukemia and hairy cell leukemiaare primary leukemic lymphomaswith a chronic course.All other non-Hodgkin lymphomascan develop secondaryleukemic diseaseChronic myelomonocytic leukemia(CMML)– Leukemic form of myelodysplasia(p.106) is classified betweenmyelodysplasia and myeloproliferativediseasesSubacute disease with transitionsinto acute forms of myeloidleukemia (secondary AML followingMDS)

Predominance of Mononuclear Round to Oval Cells91Note that in about one-fourth of all leukemias total leukocyte counts arenormal, or even reduced, and the atypical round cells affect only the relative(differential) blood analysis (“aleukemic leukemia”).In all forms of leukemia, the more fluffy layered areas of a smear show thatthe nuclear chromatin structure is not dense and coarse, as in a normallymphocyte nucleus (p. 49), but more delicately structured and irregular,often “sand-like”. A careful blood cell analysis should be carried out—perhapswith the assistance of a specialist laboratory—before bone marrowanalysis is performed.In most cases, the high leukocyte count facilitates the diagnosis ofleukemia. Apart from the leukemia-specific blast cells, a variable numberof segmented neutrophilic granulocytes may also remain, depending onthe disease progression at the time of diagnosis. This gap in the cell seriesbetween blasts and mature cells is called “leukemic hiatus.” It is found inALL but not in reactive responses or chronic myeloid leukemia, whichshow a continuous left shift. Morphological or differential diagnosis ofacute leukemia is followed by the diagnostic work-up that continues withcytochemical tests. Immunological identification of leukemia cells is alwaysindicated (Table 14).Diagnostic work-up when acute leukemia is suspected:CBC, cytochemistry, bone marrow. Collection of material to identify cellsurface markers, cytogenetics, molecular genetics.Morphological and Cytochemical Cell IdentificationAfter a first-line diagnosis of acute leukemia has been arrived at on thebasis of the cell morphology (see above), the diagnosis must be refined bycytochemical testing of blood cells or bone marrow (on fresh smears).Table 14 shows a leukemia classification based on both morphological andcytochemical criteria. The table shows that a peroxidase test allowsleukemias to be classified as peroxidase-positive (myeloid or monocytic)or peroxidase-negative (lymphoblastic). The next step is the immunologicalclassification based on cell markers.In routine clinical hematology, the FAB classification (Table 14) will bewith us for a few more years.

92Abnormalities of the White Cell SeriesTable 14 Classification of the acute nonlymphatic leukemias by morphology, cytochemistry, andimmunologyM0M1M2M3M4AML with minimalmarker differentiation,undifferentiatedblastswithout granules;distinguished fromM1 and ALL only byimmunophenotypingAML with distinct 3%marker differentiation(but without morphologicaldifferentiation);sporadic discretecytoplasmicgranulation possibleAML with morphologicallymaturecells; 10% of theblasts contain verysmall granulesAcute promyelocyticleukemia; thepredominant promyelocytescontaincopious granules,some contain Auerbodies; variant M3contains fewgranules; peripheralbilobal blastsAcute myelomonocyticleukemia;30–80% of bonemarrow blasts aremyeloblasts, promyelocytes,andmyelocytes; 20%are monocytes; variantM4 eosinophilia;additional immatureeosinophilswith dark granulesFAB type* Peroxidase PAS α-NaphthylacetatesteraseNaph-thyl-ASDesteraseImmunophenotype 3% CD 13 orCD 33 orMPO CD 79 a and cyCD 3 and cyCD 22 and CD 61/CD 41 andCD 14 3%Negativeto finegran.Negativeto finegran. MPO andCD 13/CD 33/CD 65s /and CD 14 MPO andCD 13/CD 33/CD 65s/CD 15 /and CD 14 100% MPO andCD 13 andCD 33 andnormally CD34 andHLA-DR 3% +20%+ Mixed M 1/M 2 and M 5

Predominance of Mononuclear Round to Oval Cells93Table 14ContinuedM5 a) Acute monoblasticleukemia;monoblastspredominant inthe blood andbone marrowb) Acute monocyticleukemia;monocytes inthe process ofmaturation predominanteM6M7Acute erythroidleukemia; 50% ofbone marrow blastsare erythropoietic,30% myeloblastsAcute megakaryocyticleukemia; verypolymorphic, sometimesvacuolatedblasts, some withcytoplastic blebs,sometimes aggregatedwith thrombocytes +++ 80% +++ +++FAB type* Peroxidase PAS α-NaphthylacetateesteraseNaph-thyl-ASDesteraseImmunophenotype+++ CD 13/CD 33/CD 65/CD 14/CD64 /and HLA-DR ErythroblastsGly A andCD 36 MyeloblastsMPO/CD13/CD 33/CD65s / andCD 14 CD 13/CD 33/ und CD41 oder CD61 * French American British Classification (FAB) 1976/85** A biphenotypic leukemia must be considered a possibility if several additional lymphoblasticmarkers are presentPAS Periodic acid-Schiff reaction

94Abnormalities of the White Cell SeriesChromosome analysis provides important information. In practice,however, the diagnosis of acute leukemia is still based on morphologicalcriteria.However, where the possibilities of modern therapeutic and prognosticmethods are fully accessible, new laboratory procedures based on geneticand molecular biological testing procedures form part of the diagnosticwork-up of AML. The current WHO classification takes account of thesenew methods, placing genetic, morphological, and anamnestic findings ina hierarchical order (Table 15).According to the new WHO classification, blasts account for more than20% of cells in acute myeloid leukemia (in contradistinction to myelodysplasias).Table 15WHO classification of AMLAML with specificcytogenetic translocationsAML with dysplasia inmore than 1 cell line(2 or 3 cell linesaffected)*Therapy-induced AMLund MDSAML that does not fitany of the other categories– With t(8;21) (q22; q22), AML 1/ETO– Acute promyelocytic leukemia (AML M3 with t(15;17) (q22;q11-12) and variants, PML/RAR-α– With abnormal bone marrow eosinophils and (inv16)(p13;q22) or to t(16;16) (p13; q22); CBFβ/MYH 11– With 11q23 (MLL) anomalies– With preceding myelodysplastic/myeloproliferative syndrome– Without preceding myelodysplastic syndrome– After treatment with alkylating agents– After treatment with epipodophyllotoxin– Other triggers– AML, minimal differentiation– AML without cell maturation– AML with cell maturation– Acute myelomonocytic leukemia– Acute monocytic leukemia– Acute erythroid leukemia– Acute megakaryoblastic leukemia– Acute panmyelosis with myelofibrosis– Myelosarcoma/chloroma– Acute biphenotypic leukemia*** The dysplasia must be evident in at least 50% of the bone marrow cells and in 2–3 cell lines.** Biphenotypic leukemias should be classified according to their immunophenotypes. Theyare grouped between acute lymphocytic and acute myeloid leukemias.

Predominance of Mononuclear Round to Oval Cells95Acute Myeloid Leukemias (AML)Morphological analysis makes it possible to group the predominantleukemic cells into myeloblasts and promyeloblasts, monocytes, or atypical(lympho)blasts. A morphological subclassification of these maingroups was put forward in the French–American–British (FAB) classification(Table 14).In practical, treatment-oriented terms, the most relevant factor iswhether the acute leukemia is characterized as myeloid or lymphatic.Including the very rare forms, there are at least 11 forms of myeloidleukemia.

96 Abnormalities of the White Cell SeriesAcute Myeloblastic Leukemia (Type M 0 through M 2 in the FAB Classification).Morphologically, the cell populations that dominate the CBC andbone marrow analyses (Fig. 31) more or less resemble myeloblasts in thecourse of normal granulopoiesis. Differences may be found to varyingdegrees in the form of coarser chromatin structure, more prominently definednucleoli, and relatively narrow cytoplasm. Compared with lymphocytes(micromyeloblasts), the analyzed cells may be up to threefold larger.In a good smear, the transformed cells can be distinguished from lymphaticcells by their usually reticular chromatin structure and its irregularorganization. Occasionally, the cytoplasm contains crystalloid azurophilicneedle-shaped primary granules (Auer bodies). Auer bodies (rods) areconglomerates of azurophilic granules. A few cells may begin to displaypromyelocytic granulation. Cytochemistry shows that from stage M 1 onward,more than 3% of the blasts are peroxidase-positive.Characteristics of Acute LeukemiasAge of onset: Any age.Clinical findings: Fatigue, fever, and signs of hemorrhage in laterstages.Lymph node and mediastinal tumors are typical only in ALL.Generalized involvement of all organs (sometimes including themeninges) is always present.CBC and laboratory: Hb , thrombocytes , leukocytes usuallystrongly elevated (~ 80%) but sometimes decreased or normal.In the differential blood analysis, blasts predominate (morphologiesvary).Beware: Extensive urate accumulation!Further diagnostics: Bone marrow, cytochemistry, immunocytochemistry,cytogenetics, and molecular genetics.Differential diagnosis: Transformed myeloproliferative syndrome(e.g., CML) or myelodysplastic syndrome.Leukemic non-Hodgkin lymphomas (incl. CLL).Aplastic anemias.Tumors in the bone marrow (carcinomas, but also rhabdomyosarcoma).Course, therapy: Usually rapid progression with infectious complicationsand bleeding.Immediate efficient chemotherapy in a hematology facility; bonemarrow transplant may be considered, with curative intent.

Fundamental characteristic of acute leukemia: variable blastsdrive out other cell seriesabcdFig. 31 Acute leukemia, M 0–M 2. a Undifferentiated blast with dense, fine chromatin,nucleolus (arrow), and narrow basophilic cytoplasm without granules. Thiscell type is typical of early myeloid leukemia (M 0–M 1); the final classification ismade using cell surface marker analysis (see Table 14). b The peroxidase reaction,characteristic of cells in the myeloid series, shows positive ( 3%) only for stageM 1 leukemia and higher. The image shows a weakly positive blast (1), strongly positiveeosinophil (2), and positive myelocyte (3). c and d Variants of M 2 leukemia.Some of the cells already contain granules (1) and crystal-like Auer bodies (2).97

98 Abnormalities of the White Cell SeriesAcute Promyelocytic Leukemia (FAB Classification Type M 3 and M 3 v). Thecharacteristic feature of the cells, which are usually quite large with variablystructured nuclei, is extensive promyelocytic granulation. Auer rodsare commonly present. Cytochemistry reveals a positive peroxidase reactionfor almost all cells. All other reactions are nonspecific. Acute leukemiawith predominantly bilobed nuclei is classified as a variant of M 3 (M 3 V ).The cytoplasm may appear either ungranulated (M 3 ) or very stronglygranulated (M 3 V ).Acute Myelomonocytic Leukemia (FAB Classification Type M 4). Given theclose relationship between cells in the granulopoietic and the monocytopoieticseries (see p. 3), it would not be surprising if the these two systemsshowed a common alteration in leukemic transformation. Thus, acute myelomonocyticleukemia shows increased granulocytopoiesis (up to morethan 20% myeloblasts) with altered cell morphologies, together with increasedmonocytopoiesis yielding more than 20% monoblasts or promonocytes.Immature myeloid cells (atypical myelocytes to myeloblasts)are found in peripheral blood in addition to monocyte-related cells. Cytochemically,the classification calls for more than 3% peroxidase-positiveand more than 20% esterase-positive blasts in the bone marrow. M 4 is similarto M 2 ; the difference is that in the M 4 type the monocyte series isstrongly affected. In addition to the above characteristics, the M 4Eo variantshows abnormal eosinophils with dark purple staining granules.

The diagnosis of acute leukemia is relevant even without furthersubclassificationabcedfFig. 32 Acute leukemia M 3 and M 4. a Blood analysis in promyelocytic leukemia(M 3): copious cytoplasmic granules. b In type M 3, multiple Auer bodies are oftenstacked like firewood (so-called faggot cells). c Blood analysis in variant M 3 v withdumbbell-shaped nuclei. Auer bodies d Bone marrow cytology in acute myelomonocyticleukemia M 4: in addition to myeloblasts (1) and promyelocytes (2) thereare also monocytoid cells (3). e In variant M 4Eo abnormal precursors of eosinophilswith dark granules are present. f Esterase as a marker enzyme for the monocyteseries in M 4 leukemia.99

100Abnormalities of the White Cell SeriesAcute Monocytic Leukemia (FAB Classification Types M 5 a+b). Two morphologicallydistinct forms of acute monocytic leukemias exist, monoblasticand monocytic. In the monoblastic variant M 5a , blasts predominate in theblood and bone marrow. The blast nuclei show a delicate chromatin structurewith several nucleoli. Often, only the faintly grayish-blue stained cytoplasmhints at their derivation.In monocytic leukemia (type M 5b ), the bone marrow contains promonocytes,which are similar to the blasts in monocytic leukemia, but their nucleiare polymorphic and show ridges and lobes. Some promonocytesshow faintly stained azurophilic granules. The peripheral blood containsmonocytoid cells in different stages of maturation which cannot be distinguishedwith certainty from normal monocytes. Both types are characterizedby strong positive esterase reactions in over 80% of the blasts,whereas the peroxidase reactivity is usually negative, or positive in only afew cells.Acute Erythroleukemia (FAB Classification Type M 6 )Erythroleukemia is a malignant disorder of both cell series. It is suspectedwhen mature granulocytes are virtually absent, but blasts (myeloblasts)are present in addition to nucleated erythrocyte precursors, usuallyerythroblasts (for morphology, see p. 33). The bone marrow is completelyoverwhelmed by myeloblasts and erythroblasts (more than 50% of cells inthe process of erythropoiesis). Bone marrow cytology and cytochemistryconfirm the diagnosis. Sporadically, some cases show granulopenia,erythroblasts, and severely dedifferentiated blasts, which correspond toimmature red cell precursors (proerythroblasts and macroblasts).The differential diagnosis in cases of cytopenia with red blood cell precursorsfound in the CBC must include bone marrow carcinosis, in whichthe bone marrow–blood barrier is destroyed and immature red cells (andsometimes white cells) appear in the bloodstream. Bone marrow cytologyand/or bone marrow histology clarifies the diagnosis. Hemolysis with hypersplenismcan also show this constellation of signs.Fig. 33 Acute leukemia M 5 and M 6. a In monoblastic leukemia M 5 a, blasts with a fine nuclear structure and wide cytoplasm dominate the CBC. b Seemingly maturemonocytes in monocytic leukemia M 5 b. c Homogeneous infiltration of thebone marrow by monoblasts (M 5 a). Only residual granulopoiesis (arrow). d Sameas c but after esterase staining. The stage M 5 a blasts show a clear positive reaction(red stain). There is a nonspecific-esterase (NSE)-negative promyelocyte.

Acute leukemias may also derive from monoblasts or erythroblastsabcdefFig. 33 e Same as c Only the myelocyte in the center stains peroxidase-positive(brown tint); the monoblasts are peroxidase-negative. f In acute erythrocytic leukemia(M 6) erythroblasts and myeloblasts are usually found in the blood. Thisimage of bone marrow cytology in M 6 shows increased, dysplastic erythropoiesis(e.g., 1) in addition to myeloblasts (2).101

102Abnormalities of the White Cell SeriesAcute Megakaryoblastic Leukemia (FAB Classification Type M 7 )This form of leukemia is very rare in adults and occurs more often inchildren. It can also occur as “acute myelofibrosis,” with rapid onset oftricytopenia and usually small-scale immigration into the blood of dedifferentiatedmedium-sized blasts without granules. Bone marrowharvesting is difficult because the bone marrow is very fibrous. Only bonemarrow histology and marker analysis (fluorescence-activated cellsorting, FACS) can confirm the suspected diagnosis.The differential diagnosis, especially if the spleen is very enlarged,should include the megakaryoblastic transformation of CML or osteomyelosclerosis(see pp. 112 ff.), in which blast morphology is very similar.AML with DysplasiaThe WHO classification (p. 94) gives a special place to AML with dysplasiain two to three cell series, either as primary syndrome or following a myelodysplasticsyndrome (see pp. 106) or a myeloproliferative disease (seepp. 114 ff.).Criteria for dysgranulopoiesis: 50% of all segmented neutrophilshave no granules or very few granules, or show the Pelger anomaly, or areperoxidase-negative.Criteria for dyserythropoiesis: 50% of the red cell precursor cellsdisplay one of the following anomalies: karyorrhexis, megaloblastoidtraits, more than one nucleus, nuclear fragmentation.Criteria for dysmegakaryopoiesis: 50% of at least six megakaryocytesshow one of the following anomalies: micromegakaryocytes, morethan one separate nucleus, large mononuclear cells.Hypoplastic AMLSometimes (mostly in the mild or “aleukemic” leukemias of the FAB orWHO classifications), the bone marrow is largely empty and shows only afew blasts, which usually occur in clusters. In such a case, a very detailedanalysis is essential for a differential diagnosis versus aplastic anemia (seepp. 148 f.).

New WHO classification: AML with dysplasia and hypoplasticAMLabcdFig. 34 AML with dysplasia and hypoplastic AML. a AML with dysplasia: megaloblastoid(dysplastic) erythropoiesis (1) and dysplastic granulopoiesis with Pelger-Huët forms (2) and absence of granulation in a myelocyte (3). Myeloblast (4).b Multiple separated nuclei in a megakaryocyte (1) in AML with dysplasia. Dyserythropoiesiswith karyorrhexis (2). c and d Hypoplastic AML. c Cell numbers belownormal for age in the bone marrow. d Magnification of the area indicated in c,showing predominance of undifferentiated blasts (e.g., 1).103

104Abnormalities of the White Cell SeriesAcute Lymphoblastic Leukemia (ALL)ALL are the leukemias in which the cells do not morphologically resemblemyeloblasts, promyelocytes, or monocytes, nor do they show the correspondingcytochemical pattern. Common attributes are a usually slightlysmaller cell nucleus and denser chromatin structure, the grainy consistencyof which can be made out only with optimal smear technique (i.e.,very light). The classification as ALL is based on the (often remote) similaritiesof the cells to lymphocytes or lymphoblasts from lymph nodes, andon their immunological cell marker behavior. Insufficiently close morphologicalanalysis can also result in possible confusion with chronic lymphocyticleukemia (CLL), but cell surface marker analysis (see below) will correctthis mistake. Advanced diagnostics start with peroxidase and esterasetests on fresh smears, performed in a hematology laboratory, togetherwith (as a minimum) immunological marker studies carried out on freshheparinized blood samples in a specialist laboratory. The detailed differentiationprovided by this cell surface marker analysis has prognostic implicationsand some therapeutic relevance especially for the distinction tobilineage leukemia and AML (Table 16).Table 16 Immunological classification of acute bilineage leukemias (adapted from Bene MCet al. (1995) European Group for the Immunological Characterization of Leukemias (EGIL) 9:1783–1786)Score B-lymphoid T-lymphoid Myeloid2 CytCD79a* CD3(m/cyt) MP0Cyt IgManti-TCR1 CD19 CD2 CD117CD20 CD5 CD13CD10 CD8 CD33CD10CD650.5 TdT TdT CD14CD24 CD7 CD15CD1aCD64* CD79a may also be expressed in some cases of precursor T-lymphoblastic leukemia/lymphoma.

The cells in acute lymphocytic leukemia vary, and the subtypescan be reliably identified only by immunological methodsabcdFig. 35 Acute lymphocytic leukemias. a Screening view: blasts (1) and lymphocytes(2) in ALL. Further classification of the blasts requires immunological methods(common ALL). b Same case as a . The blasts show a dense, irregular nuclearstructure and narrow cytoplasm (cf. mononucleosis, p. 69). Lymphocyte (2). c ALLblasts with indentations must be distinguished from small-cell non-Hodgkinlymphoma (e.g., mantle cell lymphoma, p. 77) by cell surface marker analysis.d Bone marrow: large, vacuolated blasts, typical of B-cell ALL. The image showsresidual dysplastic erythropoietic cells (arrow).105

106Abnormalities of the White Cell SeriesMyelodysplasia (MDS)Clinical practice has long been familiar with the scenario in which, afteryears of bone marrow insufficiency with a more or less pronounced deficitin all three cell series (tricytopenia), patients pass into a phase of insidiouslyincreasing blast counts and from there into frank leukosis—although the evolution may come to a halt at any of these stages. Thetransitions between the forms of myelodysplastic syndromes are veryfluid, and they have the following features in common:➤ Anemia, bicytopenia, or tricytopenia without known cause.➤ Dyserythropoiesis with sometimes pronounced erythrocyte anisocytosis;in the bone marrow often megaloblastoid cells and/or ring sideroblasts.➤ Dysgranulopoiesis with pseudo-Pelger-Huët nuclear anomaly (hyposegmentation)and hypogranulation (often no peroxidase reactivity) ofsegmented and band granulocytes in blood and bone marrow.➤ Dysmegakaryopoiesis with micromegakaryocytes.The FAB classification is the best-known scheme so far for organizing thedifferent forms of myelodysplasia (Table 17).Table 17Forms of myelodysplasiaForm of myelodysplasia Blood analysis Bone marrowRA = refractory anemiaRAS = refractory anemiawith ring sideroblasts( aquiredidiopathic sideroblasticanemia, p. 137)RAEB = refractory anemiawith excess ofblastsAnemia (normochromicor hyperchromic);possiblypseudo-Pelger granulocytes;blasts 1%Hypochromic andhyperchromic erythrocytesside by side,sometimes discretethrombopenia andleukopenia; pseudo-Pelger cellsOften thrombocytopeniain addition toanemia; blasts 5%,monocytes 1000/µl,pseudo-Pelger syndromeDyserythropoiesis(marginal dysgranulopoiesisand dysmegakaryopoiesis 10%) 5% blastsMore than 15% of thered cell precursors arering sideroblasts;blasts 5%Erythropoietic hyperplasia(with or withoutring sideroblasts);5–20% blastsCont. p. 108

In unexplained anemia and/or leukocytopenia and/or thrombocytopenia,blood cell abnormalities may indicate myelodysplasiaabcdeFig. 36 Myelodysplasia and CMML. a–d Different degrees of abnormal maturation(pseudo-Pelger type); the nuclear density can reach that of erythroblasts(d). The cytoplasmic hypogranulation is also observed in normal segmented granulocytes.These abnormalities are seen in myelodysplasia or after chemotherapy,among other conditions. e Blood analysis in CMML: monocytes (1), promyelocyte(2), and pseudo-Pelger cell (3). Thrombocytopenia.107

108Abnormalities of the White Cell SeriesTable 17ContinuedForm of myelodysplasia Blood analysis Bone marrowCMML = chronic myelomonocyticleukemiaRAEB in transformation(RAEB t)*Blasts 5%, monocytes 1000/µl,pseudo-Pelger syndromeSimilar to RAEB but 5% blastsHypercellular, blasts 20%, elevated promonocytesBlasts 20–30% (somecells contain Auerbodies)* In the WHO classification, the category RAEB t would belong to the category of acute myeloidleukemia.The new WHO classification of myelodysplastic syndromes defines thedifferences in cell morphology even more precisely than the FAB classification(Table 18).For the criteria of dysplasia, see page 106.The “5q- syndrome” is highlighted as a specific type of myelodysplasiain the WHO classification; in the FAB classification it would be a subtype ofRA and RAS. A macrocytic anemia, the 5q- syndrome manifests with normalor increased thrombocyte counts while the bone marrow containsmegakaryocytes with hyposegmented round nuclei (Fig. 37 b).Naturally, bone marrow analysis is of particular importance in the myelodysplasias.Table 18WHO classification of myelodysplastic syndromesDisease* Dysplasia** Blasts inperipheralbloodBlasts in thebone marrowRing sideroblastsin thebone marrowCytogenetics5q- syndrome Usually only E 5% 5% 15% 5q onlyRA Usually only DysE 1% 5% 15% VariableRARS Usually only DysE None 5% 15% VariableRCMD 2–3 lines Rarely 5% 15% VariableRCMD-RS 2–3 lines Rarely 5% 15% VariableRAEB-1 1–3 lines 5% 5–9% 15% VariableRAEB-2 1–3 lines 5–19% 10–19% 15% VariableCMML-1 1–3 lines 5% 10% 15% VariableCMML-2 1–3 lines 5–19% 10–19% 15% VariableMDS-U 1 cell lineage None 5% 15% Variable* RA = refractory anemia; RARS = refractory anemia with ring sideroblasts; RCMD = refractorycytopenia with more than one dysplastic cell line; RCMD-RC = refractory cytopenia with morethan one dysplastic lineage and ring sideroblasts; RAEB = refractory anemia with elevated blastcount; CMML = chronic myelomonocytic leukemia, persistent monocytosis (more than1 10 9 /l) in peripheral blood; MDS-U = MDS, unclassifiable. ** Dysplasia in granulopoiesis= Dys G, in erythropoiesis = DysE, in megakaryopoiesis = DysM, multilineage dysplasia= two cell lines affected; trilineage dysplasia (TLD) = all three lineage show dysplasia.

The classification of myelodysplasias requires bone marrow analysisabcdFig. 37 Bone marrow analysis in myelodysplasia. a Dysmegakaryopoiesis inmyelodysplastic syndrome (MDS). Relatively small disk-forming megakaryocytes(1) and multiple singular nuclei (2) are often seen. b Mononuclear megakaryocytes(frequent in 5q-syndrome). c Dyserythropoiesis. Particularly striking is thecoarse nuclear structure with very light gaps in the chromatin (arrow 1). Some aremegaloblast-like but coarser (arrow 2). d Iron staining of the bone marrow (Prussianblue) in myelodysplasia of the RARS type: dense iron granules forming a partialring around the nuclei (ring sideroblasts).109

110Abnormalities of the White Cell SeriesPrevalence of Polynuclear(Segmented) Cells (Table 19)Neutrophilia without Left ShiftFor clarity, conditions in which mononuclear cells (lymphocytes, monocytes)predominate were in the previous section kept distinct from hematologicalconditions in which cells with segmented nuclei and, in somecases, their precursors predominate. Leukocytosis with a predominanceof segmented neutrophilic granulocytes without the less mature forms iscalled granulocytosis or neutrophilia.Table 19 Diagnostic work-up for anomalies in the white cell series with polynucleated(segmented) cells predominatingAcute temperature,possiblyfocal signsSegmentedcells(%)Clinical findings Hb MCH LeukocytesLymphocytes(%)Other cellsn n Left shiftPatient smokesheavily (nosplenomegaly)n/ n Slowly developingfatigue,spleen /n /n Left shiftSlowly developingfatigue,spleen n// n// Normoblasts,left shift n Some normoblastsPruritus orexantheman n n/ n n EosinophilsDiagnostic steps proceed from left to right. ⎪ The next step is usually unnecessary; the next stepis obligatory.

Prevalence of Polynuclear (Segmented) Cells111Causes of Neutrophilia➤ All kinds of stress➤ Pregnancy➤ Connective tissue diseases➤ Tissue necrosis, e.g., after myocardial or pulmonary infarction➤ Acidosis of various etiologies, e.g., nephrogenic diabetes insipidus➤ Medications, drugs, or noxious chemicals, such as– Nicotine– Corticosteroids– Adrenaline– Digitalis– Allopurinol– Barbiturates– Lithium– Streptomycin– SulfonamideThrombocytesElectrophoresisTentativediagnosisn n Acute bacterialinfection(possibly withleukemoidreaction)n/ n Smoker’sleukocytosisn// n Chronicmyeloidleukemia(CML)n// n Osteomyelosclerosisn/ n Polycythemiawith concomitantleukocytosisEvidence/advanceddiagnosticsSearch for diseasefocusAnamnesisCytogenetics,BCR-ABLTear-drop-shapederythrocytesBonemarrowIn progressivedisease: bonemarrow analysis(DD myeloproliferativedisease)Completebone marrowanalysis, allfractionsOften dry tap bone marrowhistologyRef.pagep. 112p. 112p. 116p. 122p. 162n n ReactiveeosinophiliaSearch for diseasefocus/allergenp. 124

112Abnormalities of the White Cell SeriesReactive Left ShiftA relative left shift in the granulocyte series means less mature forms inexcess of 5% band neutrophils; the preceding, less differentiated cell formsare included and all transitional forms are taken into account. This leftshift almost always indicates an increase in new cell production in this cellseries. In most cases, it is associated with a raised total leukocyte count.However, since total leukocyte counts are subject to various interferingfactors that can also alter the cell distribution, left shift without leukocytosiscan occur, and has no further diagnostic value. At best, if no other explanationsoffer, a left shift without leukemia can prompt investigation forsplenomegaly, which would have prevented elevation of the leukocytecount by increased sequestration of leukocytes as in hypersplenism.In evaluating the magnitude of a left shift, the basic principle is that themore immature the cell forms, the more rarely they appear; and that thereis a continuum starting from segmented granulocytes and sometimesreaching as far as myeloblasts. Accordingly, a moderate left shift of mediummagnitude may include myelocytes and a severe left shift may go asfar as a few promyelocytes and (very rarely) myeloblasts, all depending onhow fulminant the triggering process is and how responsive the individual.The term “pathological left shift” (for a left shift that includespromyelocytes and myeloblasts) is inappropriate, because such observationscan reflect a very active physiological reaction, perhaps following a“leukemoid reaction” with pronounced left shift and leukocytosis.Causes of Reactive Left ShiftLeft shift occurs regularly in the following situations:➤ Bacterial infections (including miliary tuberculosis),➤ Nonbacterial inflammation (e.g., colitis, pancreatitis, phlebitis, andconnective tissue diseases)➤ Cell breakdown (e.g., burns, liver failure, hemolysis)Left shift sometimes occurs in the following situations:➤ Infection with fungi, mycoplasm, viruses, or parasites➤ Myocardial or pulmonary infarction➤ Metabolic changes (e.g., pregnancy, acidosis, hyperthyroidism)➤ Phases of compensation and recuperation (hemorrhages, hemolysis,or after medical or radiological immunosuppression).

Predominance of the granulocytic lineage with copious granulationand sporadic immature cells: usually a reactive left shiftabdceFig. 38 Left shift. a and b Typical blood smear after bacterial infection: toxic granulationin a segmented granulocyte (1), monocyte with gray–blue cytoplasm(2), metamyelocyte (3), and myelocyte (4). c Blood analysis in sepsis: promyelocyte(1) and orthochromatic erythroblast (2). Thrombocytopenia. d and e Reactiveleft shift as far as promyelocytes (1). Particularly striking are the reddish granulesin a band neutrophilic granulocyte (2).113

114Abnormalities of the White Cell SeriesChronic Myeloid Leukemia and MyeloproliferativeSyndrome (Chronic Myeloproliferative Disorders,CMPD)The chronic myeloproliferative disorders (previously also called the myeloproliferativesyndromes) include chronic myeloid leukemia (CML),osteomyelosclerosis (OMS), polycythemia vera (PV) and essential thrombocythemia(ET). Clearly, noxious agents of unknown etiology affect theprogenitor cells at different stages of differentiation and trigger chronicmalignant proliferation in the white cell series (CML), the red cell series(PV), and the thrombocyte series (ET). Sometimes, they lead to concomitantsynthesis of fibers (OMS). Transitional forms and mixed forms existparticularly between PV, ET, and OMS.The chronic myeloproliferative disorders encompass chronic autonomousdisorders of the bone marrow and the embryonic blood-generatingorgans (spleen and liver), which may involve one or several cell lines.The common attributes of these diseases are onset in middle age, developmentof splenomegaly, and slow disease progression (Table 20).In 95% of cases, CML shows a specific chromosome aberration(Philadelphia chromosome with a specific BCR-ABL translocation) andmay make the transition into a blast crisis.PV and ET often show similar traits (high thrombocyte count or highHb) and have a tendency to secondary bone marrow fibrosis. OMS is primarilycharacterized by fibrosis in bone marrow and splenomegaly (seep. 122).

Prevalence of Polynuclear (Segmented) Cells115Table 20 Clinical characteristics and differential diagnostic criteria in chronicmyeloproliferative diseaseSplenomegalyThrombocytesBone marrowcytologyBone marrowhistologyPhiladelphiachromosomeOther chromosomalalterationsAlkalineLeukocytephosphatase(ALP)Vitamin B 12 900 pg/mlCML(see p. 116)PV(see p. 162)OMS(see p. 122)+ No + NoLeukocytosiswith left shift,eosinophilia,basophilia !!()Very hypercellular,basophilia,megakaryocytes,andeosinophiliaGranulopoiesis, megakaryocytesLeukocytosis,hematocrit !!()Giant formsNumber ofcells Changes inthe CBC forgranulopoiesisand/orerythropoiesisTear-dropshapederythrocytes,leftshift in thegranulopoiesis,normoblastsET(see p. 170)No to () 450 000/µl !giant formsIn most casesdry tapAdvancedfibrosis !Yes! No No NoIn the accelerationphaseand blast crisisdel (20q), +8,+9, +1q, andothers-7, +8, +9,+1q, and othersMarkedlyhypercellular,erythropoiesisMegakaryocytesclearlyelevatedandarrangedin nestsMegakaryocytesclearlyelevated,arrangedin nestsVery rare ! Normal to Normal to Normal Normal

116Abnormalities of the White Cell SeriesCharacteristics of CMLAge of onset: Any age. Peak inicidence about 50 years.Clinical findings: Slowly developing fatigue, anemia; in some casespalpable splenomegaly; no fever.CBC: Leukocytosis and a left shift in the granulocyte series; possiblyHb , thrombocytes or .Advanced diagnostics: Bone marrow, cytogenetics, and moleculargenetics (Philadelphia chromosome and BCR-ABL rearrangement).Differential diagnosis: Reactive leukocytoses (alkaline phosphatase,trigger?); other myeloproliferative disorders (bone marrow, cytogenetics,alkaline phosphatase).Course, therapy: Chronic progression. Acute transformation afteryears. New, curative drugs are currently under development. Evaluatethe possibility of a bone marrow transplant (up to age approx. 60years).Steps in the Diagnosis of Chronic Myeloid LeukemiaLeft-shift leukocytosis in conjunction with usually low-grade anemia,thrombocytopenia or thrombocytosis (which often correlates with themigration of small megakaryocyte nuclei into the blood stream), and clinicalsplenomegaly is typical of CML. LDH and uric acid concentrations areelevated as a result of the increased cell turnover.The average “typical” cell composition is as follows (in a series analyzedby Spiers): about 2% myeloblasts, 3% promyelocytes, 24% myelocytes, 8%metamyelocytes, 57% band and segmented neutrophilic granulocytes, 3%basophils, 2% eosinophils, 3% lymphocytes, and 1% monocytes.In almost all cases of CML the hematopoietic cells display a markerchromosome, an anomalously configured chromosome 22 (Philadelphiachromosome). The translocation responsible for the Philadelphia chromosomecorresponds to a special fusion gene (BCR-ABL) that can be determinedby polymerase chain reaction (PCR) and fluorescence in situ hybridization(FISH).

Left shift as far as myeloblasts, proliferation of eosinophils andbasophils suggest chronic myeloid leukemia (CML)abcFig. 39 CML. a Blood analysis in chronic myeloid leukemia (chronic phase): segmentedneutrophilic granulocytes (1), band granulocyte (2) (looks like a metamyelocyteafter turning and folding of the nucleus), myelocyte with defective granulation(3), and promyelocyte (4). b and c Also chronic phase: myeloblast (1),promyelocyte (2), myelocyte with defective granulation (3), immature eosinophil(4), and basophil (5) (the granules are larger and darker, the nuclear chromatindenser than in a promyelocyte).117

118Abnormalities of the White Cell SeriesBone Marrow Analysis in CML. In many clinical situations, the findingsfrom the CBC, the BCR-ABL transformation and the enlarged spleen unequivocallypoint to a diagnosis of CML. Analysis of the bone marrowshould be performed because it provides a series of insights into the diseaseprocesses.Normally, the cell density is considerably elevated and granulopoieticcells predominate in the CBC. Cells in this series mature properly, apartfrom a slight left shift in the chronic phase of CML. CML differs from reactiveleukocytoses because there are no signs of stress, such as toxic granulationor dissociation in the nuclear maturation process.Mature neutrophils may occasionally show pseudo-Pelger forms (p. 43)and the eosinophilic and, especially, basophilic granulocyte counts areoften elevated. The proportion of cells from the red blood cell seriesdecreases. Histiocytes may store glucocerebrosides, as in Gaucher syndrome(pseudo-Gaucher cells), or lipids in the form of sea-blue precipitates(sea-blue histiocytes after Romanowsky staining).Megakaryocytes are usually increased and are often present as micromegakaryocytes,with one or two nuclei which are only slightly larger thanthose of promyelocytes. Their cytoplasm typically shows clouds ofgranules, as in the maturation of thrombocytes.

Bone marrow analysis is not obligatory in chronic myeloid leukemia,but helps to distinguish between the various chronic myeloproliferativedisordersabcFig. 40 Bone marrow cytology in CML. a Bone marrow cytology in the chronicphase: increased cell density due to increased, left-shifted granulopoiesis, e.g.,promyelocyte nest (1) and megakaryopoiesis (2). Eosinophils are increased (arrows),erythropoiesis reduced. b Often micromegakaryocytes are found in thebone marrow cytology. c Pseudo-Gaucher cells in the bone marrow in CML.119

120Abnormalities of the White Cell SeriesBlast Crisis in Chronic Myeloid LeukemiaDuring the course of CML with or without therapy, regular monitoring ofthe differential smear is particularly important, since over periods of varyingduration the relative proportions of blasts and promyelocytes increasesnoticeably. When the blast and promyelocyte fractions togethermake up 30%, and at the same time Hb has decreased to less than 10 g/dland the thrombocyte count is less than 100 000/µl, an incipient acute blastcrisis must be assumed. this blast crisis is often accompanied or precededby a markedly increased basophil count. Further blast expansion—usuallylargely recalcitrant to treatment—leads to a clinical picture not alwaysclearly distinguishable from acute leukemia. If in the chronic phase thedisease was “latent” and medical treatment was not sought, enlargementof the spleen, slight eosinophilia and basophilia, and the occasional presenceof normoblasts, together with the overwhelming myeloblast fraction,are all signs indicating CML as the cause of the blast crisis.As in AML, in two-thirds of cases cytological and immunological testsare able to identify the blasts as myeloid. In the remaining one-third ofcases, the cells carry the same markers as cells in ALL. This is a sign of dedifferentiation.A final megakaryoblastic or a final erythremic crisis is extremelyrare.Bone marrow cytology is particularly indicated when clinical symptomssuch as fatigue, fever, and painful bones suggest an acceleration of CMLwhich is not yet manifest in the CBC. In such a case, bone marrow analysiswill frequently show a much more marked shift to blasts and promyelocytesthan the CBC. A proportion of more than 20% immature cell fractionsis sufficient to diagnose a blast crisis.The prominence of other cell series (erythropoiesis, thrombopoiesis) isreduced. The basophil count may be elevated.A bone marrow aspiration may turn out to be empty (sicca) or scarcelyyield any material. This suggests fibrosis of the bone marrow, which isfrequently a complicating symptom of long-standing disease. Staining ofthe fibers will demonstrate this condition in the bone marrow histology.

In the course of chronic myeloid leukemia, an acute crisis maydevelop in which blasts predominateabcFig. 41 Acute blast crisis in CML. a Myeloblasts (1) with somewhat atypical nuclearlobes. Basophilic granulocyte (2) and band granulocyte (3). Thrombocytopenia.The proliferation of basophilic granulocytes often precedes the blast crisis.b Myeloblasts in an acute CML blast crisis. Typical sand-like chromatin structurewith nucleoli. A lymphocyte. c Bone marrow cytology in acute CML blast crisis:blasts of variable sizes around a hyperlobulated megakaryocyte (in this case duringa lymphatic blast crisis).121

122Abnormalities of the White Cell SeriesOsteomyelosclerosisWhen anemia accompanied by moderately elevated (although sometimesreduced) leukocyte counts, thrombocytopenia or thrombocytosis, clinicallyevident splenic tumor, left shift up to and including sporadic myeloblasts,and eosinophilia, the presence of a large proportion of red cell precursors(normoblasts) in the differential blood analysis, osteomyelosclerosisshould be suspected. BCR-ABL gene analysis is negative.Pathologically, osteomyelosclerosis usually originates from megakaryocyticneoplasia in the bone marrow and the embryonic hematopoieticorgans, particularly spleen and liver, accompanied by fibrosis(= sclerosis) that will eventually predominate in the surrounding tissue.The central role of cells of the megakaryocyte series is seen in the giantthrombocytes, or even small coarsely structured megakaryocyte nucleiwithout cytoplasm, that migrate into the blood stream and appear in theCBC. OMS can be a primary or secondary disease. It may arise during thecourse of other myeloproliferative diseases (often polycythemia vera oridiopathic thrombocythemia).Tough, fibrous material hampers the sampling of bone marrow material,which rarely yields individual cells. This in itself contributes to thebone marrow analysis, allowing differential diagnosis versus reactive fibroses(parainfectious, paraneoplastic).Characteristics of OMSAge of onset: Usually older than 50 years.Clinical findings: Signs of anemia, sometimes skin irritation, drasticallyenlarged spleen.CBC: Usually tricytopenia, normoblasts, and left shift.Further diagnostic procedures: Fibrous bone marrow (bone marrowhistology), when appropriate and BCR-ABL (always negative).Differential diagnosis: Splenomegaly in cases of lymphadenoma orother myeloproliferative diseases: bone marrow analysis.Myelofibrosis in patients with metastatic tumors or inflammation:absence of splenomegaly.Course, therapy: Chronic disease progression; transformation is rare.If there is splenic pressure: possibly chemotherapy, substitution therapy.Further myeloproliferative diseases are described together with the relevantcell systems: polycythemia vera (see p. 162) and essential thrombocythemia(see p. 170).

Enlarged spleen and presence of immature white cell precursorsin peripheral blood suggest osteomyelosclerosisabcdFig. 42 Osteomyelosclerosis (OMS). a and b Screening of blood cells in OMS: redcell precursors (orthochromatic erythroblast = 1 and basophilic erythroblast = 2),basophilic granulocyte (3), and teardrop cells (4). c Sometimes small, densemegakaryocyte nuclei are also found in the blood stream in myeloproliferativediseases. d Blast crisis in OMS: myeloblasts and segmented basophilic granulocytes(1).123

124Abnormalities of the White Cell SeriesElevated Eosinophil and Basophil CountsIn accordance with their physiological role, an increase in eosinophils( 400/µl, i.e. for a leukocyte count of 6000, more than 8% in the differentialblood analysis) is usually due to parasitic attack (p. 5). In the Westernhemisphere, parasitic infestations are investigated on the basis of stoolsamples and serology.Strongyloides stercoralis in particular causes strong, sometimes extreme,elevation of eosinophils (may be up to 50%). However, eosinophilia ofvariable degree is also seen in ameba infection, in lambliasis (giardiasis),schistosomiasis, filariasis, and even malaria.Bacterial and viral infections are both unlikely ever to lead to eosinophiliaexcept in a few patients with scarlet fever, mononucleosis, or infectiouslymphocytosis. The second most common group of causes of eosinophiliaare allergic conditions: these include asthma, hay fever, and various dermatoses(urticaria, psoriasis). This second group also includes druginducedhypersensitivity with its almost infinitely multifarious triggers,among which various antibiotics, gold preparations, hydantoin derivatives,phenothiazines, and dextrans appear to be the most prevalent.Eosinophilia is also seen in autoimmune diseases, especially inscleroderma and panarteritis. All neoplasias can lead to “paraneoplastic”eosinophilia, and in Hodgkin’s disease it appears to play a special role inthe pathology, although it is nevertheless not always present.A specific hypereosinophilia syndrome with extreme values (usually 40%) is seen clinically in association with various combinations ofsplenomegaly, heart defects, and pulmonary infiltration (Loeffler syndrome),and is classified somewhere between autoimmune diseases andmyeloproliferative syndromes. Of the leukemias, CML usually manifestsmoderate eosinophilia in addition to its other typical criteria (see p. 114).When moderate eosinophilia dominates the hematological picture, theterm chronic eosinophilic leukemia is used. Acute, absolute predominanceof eosinophil blasts with concomitant decrease in neutrophils, erythrocytes,and thrombocytes suggests the possibility of the very rare acuteeosinophilic leukemia.Elevated Basophil Counts. Elevation of segmented basophils to more than2–3% or 150/µl is rare and, in accordance with their physiological role inthe immune system regulation, is seen inconsistently in allergic reactionsto food, drugs, or parasites (especially filariae and schistosomes), i.e., usuallyin conditions in which eosinophilia is also seen. Infectious diseasesthat may show basophilia are tuberculosis and chickenpox; metabolic diseaseswhere basophilia may occur are myxedema and hyperlipidemia. Autonomicproliferations of basophils are part of the myeloproliferative

Eosinophilia and basophilia are usually accompanying phenomenain reactive and myeloproliferative disorders, especiallyCMLabcdFig. 43 Eosinophilia and basophilia. a Screening view of blood cells in reactiveeosinophilia: eosinophilic granulocytes (1), segmented neutrophilic granulocyte(2), and monocyte (3) (reaction to bronchial carcinoma). b and c The image showsan eosinophilic granulocyte (1) and a basophilic granulocyte (2) (clinical osteomyelosclerosis).d Bone marrow in systemic mastocytosis: tissue mast cell (3),which, in contrast to a basophilic granulocyte, has an unlobed nucleus, and the cytoplasmis wide with a tail-like extension. Tissue mast cells contain intensely basophilicgranules.125

126Abnormalities of the White Cell Seriespathologies and can develop to the extent of being termed “chronic basophilicleukemia.” In the very rare acute basophilic leukemia, the cells inthe basophilic granulocyte lineage mature in the bone marrow only to thestage of promyelocytes, giving a picture similar to type M 3 AML (p. 98).Being an expression of idiopathic disturbance of bone marrow function,elevated basophil counts are a relatively constant phenomenon in myeloproliferativesyndromes (in addition to the specific signs of these diseases),especially in CML. Acute basophilic leukemia is extremely rare; in thiscondition, some of the dedifferentiated blasts contain more or less basophilicgranules.The tissue-bound analogs of the segmented basophils, the tissue mastcells, can show benign or malignant cell proliferation, including the (extremelyrare) acute mast cell leukemia (Table 21).Table 21cellsDifferent forms of benign and malignant proliferation of tissue mastClinical picture Clinical diagnosis EvidenceIn childhood, solitary ormultiple skin nodules,some brownish Sometimes transitionDiffuse brownish papules,with urticaria on irritationHyperpigmented spots,papules and/or dermographism;histaminesymptoms: flushing, headache,pruritus, abdominalspasms, shockMalignant transformation,possibly with osteolysis,enlarged lymph nodes,splenomegaly, hepatomegalyMigration of leukemic cellsto peripheral bloodLocalized mastocytosisUrticaria pigmentosaSystemic mastocytosisMalignant mastocytosis*Acute mast cellleukemiaHistologyTypical clinicalpictureHistologyHistologyCBC (bone marrow)* May occur de novo without preceding stages and without skin involvement.

Erythrocyte and ThrombocyteAbnormalities

128Erythrocyte and Thrombocyte AbnormalitiesClinically Relevant Classification Principle for Anemias:Mean Erythrocyte Hemoglobin Content (MCH)In current diagnostic practice, erythrocyte count and hemoglobin content(grams per 100 ml) in whole blood are determined synchronously. This allowscalculation of the hemoglobin content per individual erythrocyte(mean corpuscular hemoglobin, MCH) using the following simple formula(p. 10):Hb (g/dl) · 10Ery (10 6 /µl)The mean cell volume, hematocrit, MCH, and erythrocyte size can be usedfor various calculations (Table 22; methods p. 10, normal values Table 2,p. 12). Despite this multiplicity of possible measures, however, in routinediagnostic practice the differential diagnosis in cases of low Hb concentrationor low erythrocyte counts relies above all on the MCH, and mostforms of anemia can safely be classified by reference to the normal datarange of 26–32 pg Hb/cell (1.61–1.99 fmol/cell) as normochromic (withinthe normal range), hypochromic (below the), or hyperchromic (above thenorm). The reticulocyte count (p. 11) provides important additionalpathophysiological information. Anemias with increased erythrocyte production(hyper-regenerative anemias) suggest a high reticulocyte count,while anemias with diminished erythrocyte production (hyporegenerativeanemias) have low reticulocyte counts (Table 22).It should be noted that hyporegenerative anemias due to iron or vitamindeficiency can rapidly display hyper-regeneration activity afteronly a short course of treatment with iron or vitamin supplements (up tothe desirable “reticulocyte crisis”).The practical classification of anemia starts with the MCH:— 26–32 pg = normochromic— Less than 26 pg = hypochromic— More than 32 pg = hyperchromicHypochromic AnemiasIron Deficiency AnemiaMost anemias are hypochromic. Their usual cause is iron deficiency fromvarious causes (Fig. 44). To distinguish quickly between real iron deficiencyand an iron distribution disorder, iron and ferritin levels should bedetermined.

Hypochromic Anemias129Insufficient iron absorption:Lower than normal acidity, noacidity, stomach resection,accelerated passage fromstomach to intestinesSubstances that inhibit absorptionof iron, such as citricacids and lactic acids, mucus andsimilar materialsSubstances that facilitate ironabsorption are missing(e.g. vitamin C)●●●Insufficient ironsupplementation:●●Endogenous redistribution:●Iron-deficient dietIn newborns: insufficientiron transfer from themother during gravidityTumors, infections, lunghemosiderosisChronic bleeding and pathologicalloss of iron in the following diseases:Disorders of the stomach andintestines (positive benzidinetest)Diseases of the urethra(hematuria!)Diseases of the female reproductivecycle (menorrhagia,metrorrhagia)●●●Increased iron requirement:●●Growth/maturationIncreased production of thenew blood cellsPhysiological iron lossin females:●●●MensesLactationPregnancyIron deficiency:●●●●●Fatigue, koilonychia, infectious angle(angulus infectiosus oris), Plummer-Vinson syndrome, possibly “irondeficiency fever”AnemiaHypochromy, ring-shaped erythrocytesLight serum, deficient in colorcomponentsSerum iron very reducedFig. 44 The most important reasons for iron deficiency (according toBegemann)

130Erythrocyte and Thrombocyte AbnormalitiesTable 22 Diagnostic findings and work-up for the most important disorders ofthe red cell seriesClinicalfindingsHb MCH ErythrocytemorphologyReticulocytesLeukocytesSegmentednuclei(%)Lymphocytes(%)Other cellsThrombocytesFatigue,pallor(dysphagia) Ring-shapederythrocytes,anisocytosis n n n - n/Possibly Anisocytosis,fever, weightpoikilocytosisloss n/ n n Possiblyeosinophilsmonocytes left shiftnDiffusesymptoms /n/ All sizes,dimorphic n/ n n - n/Acute n n n n () () - n//hemorrhageSubjaundice n n or pathol., n/ n n Possibly normoblastspossiblyspherocytesn/Spleen Target cells n n n Possibly normoblastsnPallerpossiblyinfectionsand bleedingtendency n n PossiblymonocytesPallorpossiblyalcohol history Macrocytes,megalocytes Possiblyhypersegmentedgranulocytes,possibly normoblasts/nPlethora,possiblyspleen n n n n/ n n - n/Acute bleeding n n () n n n - tendencyDiagnostic steps proceed from left to right. ❘ The next step is usually unnecessary; : . the nextstep is optional; the next step is obligatory. n = normal value, = lower than normal, = elevated,( ) = test not relevant, BSG = erythrocyte sedimentation rate.

Hypochromic Anemias131BSGIron FerritinandothersElectrophoresisTransferrinTentativediagnosisEvidence/further diagnosticsBone marrowRef.pagen n FerritinIron deficiencyanemiaDeterminesource of bleeding;check ironabsorptionErythropoiesis sideroblasts ,iron in macrophagesp. 132? α 2γFerritinn/Acquired/secondaryanemia triggerSearch for(infectious/toxic, paraneoplastic)n n n/ Sideroachresticanemia,myelodysplasia.Erythropoiesis ,sideroblasts iron in macrophagesp. 134. Erythropoiesis , p. 106ring sideroblastspresentn n n/ – n/ Anemia due tohemorrhagingSearch forsource andreason forhemorrhages(Erythropoiesis)p. 140n n n Haptoglobulinn n n Haptoglobulin(n/)HemolyticanemiaEspecially:thalassemia n /(n) n/ (/n) Aplastic anemiaor bonemarrow carcinosiaOsmoticresistance,Coombs test,Hb electrophoresisandfurther testsSearch for triggeror tumor.(Erythropoiesis p. 140, right shift)increased storageof ironp. 138Hypoplasia of allcell series, or carcinomacellsp. 146,148, 150n n n/ VitaminB 12folic acid()MegaloblasticanemiaGastroscopy,determinationof antibodies,possiblySchilling testn/ n Polyeythemia, ALP, progressionDD: hypergammaglobulinemiaErythropoieticmegaloblastsp. 152.Erythropoiesis p. 162n n durationofhemorrhagingPTT n– Thrombocytopenicpurpura(ITP)Search for triggers,possiblyalso for antibodiesElevated megakaryocytecountp. 166

132Erythrocyte and Thrombocyte AbnormalitiesTable 23Normal ranges for physiological iron and its transport proteinsOld unitsSI unitsSerum ironNewborns 150–200µg/dl 27–36µmol/lAdultsFemale 60–140µg/dl 11–25µmol/lMale 80–150µg/dl 14–27µmol/lTIBC 300–350µg/dl 54–63µmol/lTransferrin 250–450µg/dl 2.5–4.5 g/lSerum ferritinTIBC = Total iron binding capacity.30–300µg/l(15–160µg/l premenopausal)This usually renders determination of transferrin and total iron bindingcapacity (TIBC) unnecessary. If samples are being sent away to a laboratory,it is preferable to send serum produced by low-speed centrifugation,since the erythrocytes in whole blood can become mechanically damagedduring shipment and may then release iron. Table 23 shows the variationof serum iron values according to gender and age.It is important to note that acute blood loss causes normochromic anemia.Only chronic bleeding or earlier serious acute blood loss leads to irondeficiency manifested as hypochromic anemia.Iron Deficiency and Blood Cell Analysis Focusing on the erythrocyte morphologyis the quickest and most efficient way to investigate hypochromicanemia when the serum iron has dropped below normal values. In hypochromicanemia with iron and hemoglobin deficiency (whether due to insufficientiron intake or an increased physiological iron requirement),erythrocyte size and shape does not usually vary much (see Fig. 45). Only inadvanced anemias (from approx. 11 g/dl, equivalent to 6.27 mmol/l Hb)are relatively small erythrocytes (microcytes) with reduced MCV andMCH and grayish stained basophilic erythrocytes (polychromatic erythrocytes)seen, indicating inadequate hemoglobin content. Cells with theappearance of relatively large polychromatic erythrocytes are reticulocytes.The details of their morphology can be seen after supravital staining(p. 141). A few target cells (p. 139) will be seen in conditions of severe irondeficiency.In severe hemoglobin deficiency ( 8 g/dl, equivalent to 4.96 mmol/l)the residual hemoglobin is found mostly at the peripheral edge of theerythrocyte, giving the appearance of a ring-shaped erythrocyte.

Small, hemoglobin-poor erythrocytes indicate iron deficiencyabcdFig. 45 Iron deficiency anemia. a and b Erythrocyte morphology in iron deficiencyanemia: ring-shaped erythrocytes (1), microcytes (2) faintly visible target cells(3), and a lymphocyte (4) for size comparison. Normal-sized erythrocytes (5) aftertransfusion. c Bone marrow cytology in iron deficiency anemia shows only increasedhematopoiesis and left shift to basophilic erythroblasts (1). d Absence ofiron deposits after iron staining (Prussian blue reaction). Megakaryocyte (1).133

134 Erythrocyte and Thrombocyte AbnormalitiesHypochromic Infectious or Toxic Anemia(Secondary Anemia)Among the various causes of lack of iron for erythropoiesis (see Fig. 44,p. 129), a special situation is represented by the internal iron shift causedby “iron pull” of the reticuloendothelial system (RES) during infections,toxic processes, autoimmune diseases, and tumors. Since this anemia resultsfrom another disorder, it is also called secondary anemia. The MCH ishypochromic, or in rare instances, normochromic, and therefore erythrocytemorphology is particularly important to diagnosis. In contrast to exogenousiron deficiency anemias, the following phenomena are often observed,depending on the severity of the underlying condition:➤ Anisocytosis, i.e., strong variations in the size of the erythrocytes, beyondthe normal distribution. The result is that in almost every fieldview, some erythrocytes are either half the size or twice the size of theirneighbors.➤ Poikilocytosis, i.e., variations in the shape of the erythrocytes. In additionto the normal round shape, numbers of oval, or pear, or tear shapedcells are seen.➤ Polychromophilia, the third phenomenon in this series of nonspecificindicators of disturbed erythrocyte maturation, refers to light graybluestaining of the erythrocytes, indicating severely diminishedhemoglobin content of these immature cells.➤ Basophilic cytoplasmic stippling in erythrocytes is a sign of irregular regenerationand often occurs nonspecifically in secondary anemia.The reticulocyte count is usually reduced in infectious or toxic anemia, unlessthere is concomitant hemolysis or acute blood loss.Bone marrow analysis in secondary anemia usually shows reducederythropoiesis and granulopoiesis with a spectrum of immature cells (“infectious/toxicbone marrow”). The information is so nonspecific that usuallybone marrow aspiration is not performed. So long as all other laboratorymethods are employed, bone marrow cytology is very rarely neededin cases of hypochromic anemia.

Hypochromic erythrocytes of very variable morphology indicatesecondary anemia, usually in cases of infectious disease ortumorabcFig. 46 Secondary anemia. a and b Erythrocyte morphology in secondary hypochromicanemia: the erythrocytes vary greatly in size (anisocytosis) and shape (1)(poikilocytosis), and show basophilic stippling (2). Burr cell (3), which has no specificdiagnostic significance. Occasionally, the erythrocytes stain a soft gray–blue(4) (polychromasia). c Bone marrow cell overview in secondary anemia. Cellcounts in the white cell series are elevated (promyelocytes = 1), eosinophils (2),and plasma cells (3); erythropoiesis is reduced (4).135

136 Erythrocyte and Thrombocyte AbnormalitiesBone Marrow Cytology in the Diagnosis of HypochromicAnemiasSo long as all other laboratory methods are employed, bone marrow cytologyis very rarely needed in cases of hypochromic anemia.Bone marrow cytology is rarely strictly indicated after all other availablediagnostic methods have been exhausted (Table 22, p. 130). However, indoubtful cases it can usually at least help to rule out malignant disease.In iron deficiency anemias of the most various etiologies, erythropoiesisis stimulated in a compensatory fashion, and the distribution of themarkers shows the expected increase in red cell precursors. The erythropoiesisto granulopoiesis ratio increases in favor of erythropoiesis from1 :3 to 1 :2, but rarely further. The red cell series shows a left shift, i.e.,there are more immature red cell precursors (erythroblasts and proerythroblasts)(for normal values, see Table 4). Usually, these red cell precursorsdo not show any clearly atypical morphology, but the cytoplasm is basophiliceven in normoblasts, according with the poor hemoglobinization.Iron staining of the bone marrow shows no sideroblasts (normoblasts containingiron granules), or only very few ( 10%, norm 30–40%). A constantfinding in anemia stemming from exogenous iron deficiency is absence ofiron in the macrophages of the bone marrow reticulum.Megakaryocyte counts are almost always increased in iron deficiencydue to chronic hemorrhaging, but can also show increased proliferation iniron deficiency from other causes (which can lead to increased thrombocytecounts in states of iron deficiency).In infectious or toxic (secondary) anemia, unlike exogenous iron deficiencyanemia, erythropoiesis tends to be somewhat suppressed. There isno left shift and no specific anomalies are present. Granulopoiesis predominatesand often shows nonspecific “stress phenomena” and a dissociationof nuclear and cytoplasmic maturation (e.g., cytoplasm that is stillbasophilic with promyelocytic granules in mature, banded myelocyte nuclei).Depending on the trigger of the anemia, the monocyte, lymphocyte, orplasma cell counts are often moderately increased, and megakaryocytecounts are occasionally slightly elevated. The important indicator is ironstaining of the bone marrow. The “iron pull” of the RES leads to intensiveiron storage in macrophages, while the red cell precursors are almost ironfree.However, combinations do exist, when a pre-existing iron deficiencymeans that the iron depositories are empty even in an infectious or toxicprocess. Moreover, not every secondary anemia is hypochromic. Wherethere is concomitant alcoholism or vitamin deficiency, secondary anemiamay be normochromic or hyperchromic.

Hypochromic Anemias137Hypochromic Sideroachrestic Anemias(Sometimes Normochromic or Hyperchromic)In a sideroachrestic anemia existing iron cannot be utilized (achrestic= useless). This is a suspected diagnosis when serum iron levels areraised or in the high normal range, and when the erythrocytes show stronganisocytosis, poikilocytosis (with reduced average MCV), polychromophilia,and in some cases also basophilic stippling (Fig. 46). This suspicion can befurther illuminated by bone marrow analysis. Unlike in infectious/toxicanemias, the red cell series is well represented. Iron staining of the bonemarrow is the decisive diagnostic test, causing the iron-containing redprecursor cells (sideroblasts) to stand out (hence the term “sideroblasticanemia.”) The iron precipitates often collect in a ring around the nucleus(“ringed sideroblasts”).By far the majority of the “idiopathic sideroachrestic anemias,” as theyused to be called, are myelodysplasias (see p. 106). Only a few of them appearto be hereditary or have exogenous triggers (alcoholism, lead poisoning).

138 Erythrocyte and Thrombocyte AbnormalitiesHypochromic Anemia with HemolysisThalassemiasA special form of hypochromic anemia mostly affecting patients of Mediterraneandescent presents with normal erythrocyte count, decreasedMCH, and clinical splenomegaly. The smear displays erythrocytes withcentral hemoglobin islands (target cells). These cells do not necessarilypredominate in the CBC: the most revealing field views show at most 50%target cells in addition to clear anisocytosis and frequent basophilic stippling.Occasional normoblasts give a general indication of increased erythropoiesis.Although target cells are also nonspecific, since they can occurin such conditions as severe iron deficiency or obstructive jaundice, thisoverall picture should prompt hemoglobin electrophoresis. The sampleconsists of ACD-stabilized blood at 1 :10 dilution. A significant increase inthe HbA 2 fraction confirms a diagnosis of thalassemia minor, the heterozygousform of the disease. Thalassemia major, the homozygous variant, isfar rarer and more serious. In this form of the disease, in addition to thetarget cells, the CBC shows a marked increase in red precursor cells. Hbelectrophoresisshows a predominance of HbF (the other hemolytic anemiasare usually normochromic, see p. 140).

Hypochromic anemia without iron deficiency, sometimes withtarget cells, suggests thalassemiaabcFig. 47 Thalassemia. a Thalassemia minor: often no target cells, but an increasein the number of small erythrocytes (shown here in comparison with a lymphocyte),so that sometimes there is no anemia. b More advanced thalassemia minor:strong anisocytosis and poikilocytosis (1), basophilic stippling (2), and sporadictarget cells (3). c Thalassemia major: erythroblasts (1), target cell (2), polychromaticerythrocytes (3), and Howell–Jolly bodies (4) (in a case of functional asplenia).Lymphocyte (5) and granulocyte (6).139

140 Erythrocyte and Thrombocyte AbnormalitiesNormochromic AnemiasAnemias where red cell hemoglobinization is normal (26–32 pg/dl, equivalentto 1.61–1.99 fmol/l) and average MCVs are normal (77–100 fl) canbroadly be explained by three mechanisms: a) acute blood loss with sufficientmetabolic reserves remaining; b) elevated cell turnover in which ironis reused as soon as it becomes free, so that hypochromia does not arise(this is typical of almost all hemolytic anemias except thalassemias; seep. 138); and c) suppression of cell production under conditions of normaliron supply (this is the group of hypoplastic–aplastic anemias, which havea variety of causes).— In cases of acute blood loss: clinical findings, occult blood?— In hemolytic cases: reticulocytes , haptoglobin , possibly bilirubin.— In bone marrow suppression: e.g. aplastic anemia, reticulocytes .Normochromic Hemolytic AnemiasHemolytic anemias result from a shortened erythrocyte life span with insufficientcompensation from increased erythrocyte production (Table24).Usually, hematopoiesis in the bone marrow is increased in compensation,and, depending on the course of the disease, may make up for the acceleratedcell degradation for all or some of the time by recycling the iron asit becomes free.Accordingly, counts of the young, newly emerged erythrocytes (reticulocytes)are always raised, and usually sporadic normoblasts are found. Anemiaproper often becomes apparent only in a “crisis” with acute, acceleratedcell degradation, and reticulocyte counts increased up to more than500%.A common cellular phenomenon after extended duration of hemolyticanemia is the manifestation of macrocytic hypochromic disorders (p. 150),because the chronic elevation of hematopoietic activity can exhaust theendogenous folic acid reserves (pernicious anemia).Bone marrow analysis shows both relative and absolute increases inerythropoietic activity: among the red cell precursors, in acute severehemolysis the more immature forms often predominate more than in normalbone marrow, and in chronic hemolysis the maturer forms do(orthochromatic normoblasts). In addition, the normoblasts in hemolyticbone marrow often are markedly clustered (Fig. 48), whereas in normalbone marrow they are more evenly dispersed (Fig. 18).

Consistently elevated “young” erythrocytes (reticulocytes) suggesthemolysisabcFig. 48 Hemolytic anemia. a and b Newly formed erythrocytes appear as large,polychromatic erythrocytes (1) after Pappenheim staining (a); supravital staining(b) reveals spot-like precipitates (reticulocyte = 2). Thrombocyte (3). c Bone marrowcells in hemolytic anemia at low magnification: increased hematopoiesis withcell clusters. Orthochromatic erythroblasts predominate. A basophilic erythroblastshows loosened nuclear structure (arrow), a sign of secondary folic acid deficiency.141

142Erythrocyte and Thrombocyte AbnormalitiesTable 24Causes of the most common hemolytic anemiasSpecial morphologicalfeatures of erythrocytesCauses within the erythrocytes (corpuscular hemolyses)➤➤Hereditary Membrane abnormalities– Spherocytosis (see p. 144)– Elliptocytosis Hemoglobin abnormalities– Thalassemia (see p. 138)– Sickle cell anemia (seep. 144)– Other rare hemoglobinrelateddisorders Enzyme defects– Glucose-6-phosphatedehydrogenase– Pyruvate kinase andmany othersAcquired Paroxysmal nocturnalhemoglobinuria Zieve syndrome– Small spherocytes– Elliptocytes– Target cells– Sickle cells– Possibly Heinz bodies– Macrocytes– Foam cells in the bonemarrowFurther advanceddiagnosticsOsmotic resistanceHemoglobin electrophoresisEnzyme testsSucrose hemolysistest, absence of CD 55(DAF) and CD 59MIRL (membraneinhibitor of reactivelysis)Causes outside the erythrocytes (extracorpuscular hemolyses)➤ Biosynthesis of antibodies Isoantibodies (fetal erythroblastosis,Rh serologytransfusion events) Warm autoantibodiesCoombs test Cold autoantibodies – Autoagglutination Coombs test,Cold agglutinationtiter Chemical-allergic antibodies(e. g., cephalosporin, methyldopa)➤ Physical or chemical noxae(e. g., after burns, heart valvereplacement; heavy metalexposure, animal- or plantderivedpoisons)– Partially Heinz bodies➤ Microangiopathic hemolysis inhemolytic-uremic syndrome,thrombotic-thrombocytopenicpurpura, bone marrow carcinoses➤ Infection-related noxae (e. g.influenza, salmonella infection,malaria)➤Hypersplenism, e. g. lymphaticsystem disease, infections withsplenomegaly, portal hypertension– Schizocytes, fragmentocytes(see p. 143)– For malaria pathogen(see p. 158)Thrombocytes Liver, kidneyDemonstration ofpathogenCause ofsplenomegaly

Distribution pattern and shape of erythrocytes can be relevant inthe diagnosis of hemolysisabcFig. 49 Autoagglutination and fragmentocytes. a Clumps of erythrocytes. If thisis the picture in all regions of the smear, an artifact is unlikely and serogenic (auto)agglutinationshould be suspected (in this case due to cryoagglutinins in mycoplasmicpneumonia). Thrombocytes are found between the agglutinated erythrocytes.b and c Conspicuous half-moon and egg-shell-shaped erythrocytes: fragmentocytosisin microangiopathic hemolytic anemia. Fragmentocytes (1), targetcell (2), and echinocytes (3) (this last has no diagnostic relevance).143

144 Erythrocyte and Thrombocyte AbnormalitiesCytomorphological Anemias with ErythrocyteAnomaliesMicrospherocytosis This corpuscular form of hemolysis is characterizedby dominant genetic transmission, splenomegaly, and a long uneventfulcourse with occasional hemolytic crises. Blood analysis shows erythrocyteswhich appear strikingly small in comparison with leukocytes. Thecentral light area is absent or only faintly visible, since they are sphericalin cross-section rather than barbell-shaped. The abnormal size distributioncan be measured in two dimensions and plotted using Price–Jonescharts. Close observation of the morphology in the smear (Fig. 50) is particularlyimportant, because automated blood analyzers will determinea normal cell volume. The extremely reduced osmotic resistance of theerythrocytes (in the NaCl dilution series) is diagnostic. Coombs test isnegative.Stomatocytosis is an extremely rare hereditary condition. Stomatocytesare red cells with a median streak of pallor, giving the cells a “fish mouth”appearance. A few stomatocytes may be found in hepatic disease.Hemoglobinopathies (see also thalassemia, p. 138). Target cells arefrequently present (Fig. 47).In addition to target cells, smears from patients with sickle cell anemiamay show a few sickle-shaped erythrocytes, but more usually these onlyappear under conditions of oxygen deprivation (Fig. 50). (This can beachieved by covering a fresh blood droplet with a cover glass; a droplet of2% Na 2 S 2 O 4 may be added). Sickle cell anemia is a dominant autosomal recessivehemoglobinopathy and is diagnosed by demonstrating the HbSband in Hb-electrophoresis. Homozygous patients with sickle cell anemiaalways suffer from chronic normocytic hemolysis. If provoked by oxygendeficiency or infections, severe crises may occur with clogging of the microvasculatureby aggregates of malformed erythrocytes. Patients who areheterozygous for sickle cell anemia have the sickle cell trait but do not displaythe disease or its symptoms. These can, however, be triggered by verylow oxygen tension. Sickle cell anemia is quite often combined with otherhemoglobinopathies, such as thalassemia.Schistocytosis (Fragmentocytosis) If, in acquired hemolytic anemia, someof the erythrocytes are fragmented and have various irregular shapes(eggshell, helmet, triangle, or crescent; Fig. 49), this may be an indicationof changes in the capillary system (microangiopathy), or else of disseminatedintravascular coagulopathy (DIC). Microangiopathic hemolytic anemiasdevelop in the course of thrombotic thrombocytopenic purpura (TTP,Moschcowitz disease) and its related syndromes: in children (hemolyticuremic syndrome, HUS); in pregnant women (HELLP syndrome); or inpatients with bone marrow metastases from solid tumors.

Conspicuous erythrocyte morphology in anemia: microspherocytosisand sickle cell anemiaabcdFig. 50 Microspherocytes and sickle cells. All erythrocytes are strikingly small incomparison with lymphocytes (1) and lack a lighter center: these are microspherocytes(diameter 6 µm). Polychromatic erythrocyte (2). b Erythrocytes with anelongated rather than round lighter center: these are stomatocytes, which are rarelythe cause of anemia. c Native sickle cells (1) are found only in homozygoussickle cell anemia, otherwise only target cells (2) are present. d Sickle cell test underreduced oxygen tension: almost all erythrocytes appear as sickle cells in thehomozygous case presented here.145

146 Erythrocyte and Thrombocyte AbnormalitiesNormochromic Renal Anemia(Sometimes Hypochromic or Hyperchromic)Normochromic anemia should also suggest the possibility of renal insufficiency,which will always lead to anemia within a few weeks. In cases ofchronic renal insufficiency it is always present and may reach Hb values aslow as 6 g/dl. The anemia is caused by changes in the synthesis of erythropoietin,the hormone regulating erythropoiesis; measurement of serumerythropoietin is an important diagnostic tool. Erythrocyte life span is alsoslightly reduced.Apart from poikilocytosis, the blood cells are morphologically unremarkable.The reticulocyte counts often remain normal. The bone marrowdoes not show any significant characteristic changes, and therefore servesno diagnostic purpose in this situation.Anemias due to renal insufficiency are usually normochromic, but hypochromicor hyperchromic forms do occur. Hypochromic anemia is an indicatorof the reactive process that has led to the renal insufficiency (e.g.,pyelonephritis and glomerulonephritis), resulting in secondary hypochromicanemia. In addition, dialysis patients often develop iron deficiency.Chronic renal insufficiency can lead to folic acid deficiency, anddialysis therapy will reinforce this, explaining why hyperchromic anemiasalso occur in kidney disease.Bone Marrow AplasiaPure Red Cell Aplasia (PRCA, Erythroblastopenia)Erythroblastopenia in the sense of a purely aplastic condition in the redcell series is extremely rare. Diamond–Blackfan anemia (congenital hypoplasticanemia) is the congenital form of this disease. Acquired acute, transientinfections in adults and children are usually caused by virus infections(parvovirus B19). Chronic acquired erythroblastophthisis isfrequently associated with thymoma and has an autoimmune etiology.Anemia in pure erythroblastophthisis is normochromic without significantchanges in the CBC for white cells and thrombocytes. Naturally, thereticulocyte count is extremely low, close to zero.In all these anemias, the bone marrow shows well-developed granulopoiesisand megakaryopoiesis, but erythropoiesis is (more or less) entirelylacking.

Unexplained decrease in cell counts for one or more lines: thebone marrow smear may show various forms of aplasiaabcdFig. 51 Forms of bone marrow aplasia. a Bone marrow cytology in erythroblastopenia:only activated cells of the granulopoietic series are present. The megakaryopoiesis(not shown here) show no abnormalities. b Bone marrow aplasia:hematopoiesis is completely absent: only adipocytes and stroma cells are seen.c Giant erythroblast (arrow) in the bone marrow in acute parvovirus B19 infection.d Conspicuous binuclear erythroblasts in the bone marrow of a patient with congenitaldyserythropoietic anemia (type II CDA).147

148Erythrocyte and Thrombocyte AbnormalitiesThe differential diagnosis in this context relates to very rare congenitaldyserythropoietic anemias (CDA). These anemias manifest mostly inchildhood or youth and may be normocytic or macrocytic. The bone marrowshows increased erythropoiesis with multinucleated erythroblasts,nuclear fragmentation, and cytoplasmic bridges. There are three types(type II carries the so-called HEMPAS antigen: hereditary multinuclearitywith positive acidified serum lysis test).Aplasias of All Bone Marrow Series (Panmyelopathy,Panmyelophthisis, Aplastic Anemia)A reduction in erythrocyte, granulocyte, and thrombocyte cell counts inthese series, which may progress to zero, is far more common than pureerythroblastopenia and is always acquired (except in the rare pediatricFanconi syndrome with obvious deformities).Pathologically, this life-threatening disease is a result of damage to thehematopoietic stem cells, often by chemical toxins or, occasionally, viralinfection. An autoimmune response of the T-lymphocytes also seems toplay a role.The term “panmyelopathy” is synonymous with “aplastic anemia” inthe broader sense and with “panmyelophthisis.”The CBCs show the rapid progression of normochromic anemia andgreatly reduced reticulocyte counts. Granulocyte counts graduallydwindle to zero, followed by the monocytes. Thrombocytes are usuallyalso quite severely affected.The remaining blood cells in all series appear normal, although naturally,given the presence of the noxious agents or intercurrent infections,they often show reactive changes (e.g., toxic granulations). The bone marrowaspirate for cytological analysis is often notable for poor yield ofmaterial, although a completely empty aspirate (dry tap) is rare.The material obtained yields unfamiliar images in a smear (Fig. 51).Frequently, strings and patches of reticular (stroma) cells from the bonemarrow predominate, which normally are barely noticed in an aspirate.There are usually no signs of phagocytosis. Aside from the reticular cells,there are isolated lymphocytes, plasma cells, tissue basophils, and macrophages.Depending on the stage in the aplastic process, there may be residualhematopoietic cells. In some instances, the whole disease process isfocal. For this reason, bone marrow histology must be performedwhenever the cytological findings are insufficient or dubious in cases oftricytopenia of unknown cause.

Hypochromic Anemias149Differential Diagnosis versus Reduction in Cell Counts in Several Series(Bicytopenia or Tricytopenia):➤ After thorough analysis, most cytopenias with hyperplasia of the bonemarrow have to be defined as myelodysplasias (p. 106), unless they arecaused by accelerated cell degeneration (e.g., in hypersplenism).➤ Cytopenia with bone marrow fibrosis points to myeloproliferative-typediseases (p. 114) or results from direct toxic or inflammatory agents.➤ Cytopenia can of course also occur after bone marrow infiltration bymalignant cells (carcinoma, sarcoma), which will not be contained inevery bone marrow aspirate. This is why, when the diagnosis is uncertain,histological analysis should always be carried out.➤ Cytopenia can also result from B 12 or folic acid deficiency (but note thepossibility of hyperchromic anemia, p. 152).➤ Another cause of cytopenia is expansion of malignant hematopoieticcells in the bone marrow. This is easily overlooked if the malignant cellsdo not appear in the bloodstream, as in plasmacytoma, lymphadenoma,and aleukemic leukemia (leukemia without peripheral blasts).➤ Cytopenia also develops of course after high-dose radiation orchemotherapy. In such cases it is not so much the CBC or bone marrowanalysis as the exposure history that will allow the condition to be distinguishedfrom the panmyelopathies described above.➤ Cytopenia caused by panmyelopathy mechanisms (see preceding text;triggers shown in Table 25).Table 25Substances, suspected or proven to cause panmyelopathyAnalgesics, antirheumaticdrugsAntibioticsAnticonvulsive drugsThyrostatic drugsSedativesOther medicationsInsecticidesSolventsVirusesPhenylbutazone, oxyphenbutazone, othernonsteroidal antirheumatic drugs, goldpreparations, penicillamineChloramphenicol, sulfonamideHydantoinCarbimazole/methimazolePhenothiazinesCimetidine, tolbutamideHexachlorcyclohexane and other chlorinatedhydrocarbonsBenzenez. B. Hepatitis, CMV

150Erythrocyte and Thrombocyte AbnormalitiesIn unexplained anemia, thrombocytopenia, or leukocytopenia, anypossible triggers (Table 25) must be discontinued or avoided. Panmyelophthisisor aplastic anemia is an acute disease that can only be overcomewith aggressive treatment (glucocorticoids, cyclosporin, antilymphocyteglobulin).Bone Marrow Carcinosis and Other Space-OccupyingProcessesAnemia resulting from bone marrow infiltration by growing, spaceoccupyingtumor metastases can in principle be normochromic. However,under the indirect influence of the underlying disease, it tends more oftento be hypochromic (secondary anemia).Normoblasts in the differential blood analysis (Fig. 9 a, p. 33) particularlysuggest the possibility of bone marrow carcinosis, because their presenceimplies destruction of the bone marrow–blood barrier. Usually, bone marrowcarcinosis leads eventually to lower counts in other cell series,especially thrombocytes.Bone metastases from malignant tumors rarely affect the bone marrowand hematopoiesis, and if they do, it is usually late. The most commonmetastases in bone marrow derive from small-cell bronchial carcinomaand breast cancer.In the differential diagnosis, the effects of direct bone marrow infiltrationmust be distinguished from phenomena caused by microangiopathichemolytic anemia (MHA) in the presence of tumor (p. 144). Carcinosis andMHA may of course coexist.Bone marrow cytology in these situations tends to reveal a generallydecreased density of hematopoietic cells and signs of reactive marrow asseen in secondary anemia (p. 134). Only in a few field views—often at theedge of the smear—will one occasionally encounter atypical cell elementswhich cannot be assigned with certainty to any of the hematopoietic blastfamilies. The critical feature is their close arrangement in clusters. Theseatypical cells are at least as large as myeloblasts or proerythroblasts (e.g.,in small-cell bronchial carcinoma), usually considerably larger. Tumortype cannot be diagnosed with certainty (except, e.g., melanoma). Bonemarrow histology and possibly immunohistology tests must be performedif there is any doubt, or in the case of negative cytological findingsor dry tap, since the clustered, focal character of metastases naturallymeans that they may not be obtained in every aspirate.

Thrombocytopenia with leukocytosis and erythroblasts in theperipheral blood: consider bone marrow carcinosisabcFig. 52 Bone marrow carcinosis. a and b Bone marrow smear at low magnificationshowing islands of infiltration by a homogeneous cell type (a), or, alternatively,by apparently different cell types which do, however, all display identical chromatinstructure and cytoplasm: bone marrow carcinosis in breast carcinoma (a) orbronchial non-small-cell carcinoma (b). c Island of dedifferentiated cells in thebone marrow which cannot be assigned to any of the hematopoietic lineages:bone marrow carcinosis (here in a case of embryonal testicular cancer).151

152Erythrocyte and Thrombocyte AbnormalitiesHyperchromic AnemiasIn patients with clear signs of anemia, e.g., a “sickly pallor,” atrophic lingualmucosa, and sometimes also neurological signs of bathyanesthesia(loss of deep sensibility), even just a cursory examination of the bloodsmear may indicate the diagnosis. Marked poikilocytosis and anisocytosisare seen, and the large size of the erythrocytes is particularly conspicuousin comparison with the lymphocytes, whose diameter they exceed (megalocytes).These are the hallmarks of macrocytic, and, with respect to bonemarrow cells, usually also megaloblastic anemia, with a mean cell diametergreater than 8 µm and a cell volume (MCV) usually greater than100 µm 3 . Mean cell Hb content (MCH) is more than 36 pg (1.99 fmol) andthus indicates hyperchromic anemia.Only when there is severe pre-existing concomitant iron deficiency is acombination of macrocytic cells and hypochromic MCH possible (“dimorphicanemia”).Table 26Most common causes of hyperchromic anemiasVitamin-B 12 deficiencyNutritional deficits, e. g.– Goat milk– Vegetarian diet– AlcoholismImpaired absorption– Genuine pernicious anemia– Status after gastrectomy– Ileum resection– Crohn disease– Celiac disease, sprue (psilosis)– Intestinal diverticulosis– Insufficiency of the exocrine pancreas– Fish tapewormFolic acid deficiencyNutritional deficits– Chronic abuse of alcoholImpaired absorption– E.g., sprue (psilosis)Increased requirement– Pregnancy– Hemolytic anemiaInterference/antagonism– Phenylhydantoin– Cytostatic antimetabolic drugs– Trimethoprim (antibacterialcombination drug)– Oral contrazeptives– Antidepressants– AlcoholAlthough other rare causes exist (Table 26), almost all patients with hyperchromicanemia suffer from vitamin B 12 and/or folic acid deficiency.Since a deficiency of these essential metabolic building blocks suppressesDNA synthesis not only in erythropoiesis, but in the other cell series aswell, over time more or less severe pancytopenia will develop.

Conspicuous large erythrocytes suggest hyperchromic macrocyticanemia, usually megaloblastic in the bone marrowabcdFig. 53 Hyperchromic anemia. a Marked anisocytosis. In addition to normalsizederythrocytes (1), macrocytes (2) and large ovoid megalocytes are seen (3).Hypersegmented granulocyte (4). b In hyperchromic anemia, red cell precursorsmay be released into the peripheral blood: here, a polychromatic erythroblast. cand d Bone marrow in megaloblastic anemia: slight (1) or marked (2) looseningup of the nuclear structure, in some cases with binuclearity (3). Giant forms ofband granulocytes and metamyelocytes (4) are often present.153

154Erythrocyte and Thrombocyte AbnormalitiesWith hypersegmentation, i.e. 4–5 segments/nucleus, the segmentedgranulocytes show all the indications of a maturation disorder. In addition,the reticulocyte count is increased (but it may also be normal), andthe iron content is elevated or normal.Megaloblastic anemias show the same hematological picture whetherthey are caused by folic acid deficiency or by vitamin B 12 deficiency.Table 26 lists possible causes. It should be emphasized that “genuine” perniciousanemia is less common than megaloblastic anemia due to vitaminB 12 deficiency. In pernicious anemia a stomach biopsy shows atrophicgastritis and usually also serum antibodies to parietal cells and intrinsicfactor.Among the causes of folic acid deficiency is chronic alcoholism (withinsufficient dietary folic acid, impaired absorption, and elevated erythrocyteturnover). On the other hand, many alcoholics with normal vitaminB 12 and folic acid levels develop severe hyperchromic anemia with aspecial bone marrow morphology, obviously with a pathomechanism ofits own (pyridoxine [B 6 ] deficiency, among others).In megaloblastic anemia (Fig. 53) the cell density in the bone marrow isalways remarkably high. Large to medium-sized blasts with round nucleidominate the erythrocyte series. They are present in varying sizes, theirchromatin is loosely arranged with a coarse “sandy” reticular structure,there are well-defined nucleoli, and the cytoplasm is very basophilic witha perinuclear lighter zone. These cells can be interpreted as proerythroblastsand macroblasts whose maturation has been disturbed. As thesedisturbed megaloblastic cells appear along a continuous spectrum fromthe less mature to the more mature, they are all referred to collectively asmegaloblasts.In the granylocytic series, anomalies become obvious at the myelocytestage; characteristic giant cells with loosely structured nuclei developwhich may tend to be classified as myelocytes/stab cells, but which in factprobably are myelocytes in which the maturation process has been disturbed.As in peripheral blood smears, segmented granulocytes are oftenhypersegmented. Megakaryocytes also show hypersegmentation of theirnuclei or many individual nuclei. Iron staining reveals increased numberof iron-containing reticular cells and sideroblasts, and a few ring sideroblastsmay develop. All these changes disappear after vitamin B 12 supplementation,after just three days in the erythrocyte series and withinone week in the granulocyte series. In the differential diagnosis, in relationto the causes listed in Table 26, the following should be highlighted: toxicalcohol damage (vacuolized proerythroblasts), hemolytic anemia (elevatedreticulocyte count), myelodysplasia (for bone marrow morphology seeFig. 37, p. 109).

In older patients, myelodysplastic syndrome should be the firstitem in the differential diagnosis of hyperchromic anemiasabcFig. 54 Myelodysplastic syndrome (MDS) as differential diagnosis in hyperchromicanemia. a Strongly basophilic stippling in the cytoplasm of a macrocyte(in myelodysplasia). b Myeloblast with hyperchromic erythrocyte as an exampleof a myelodysplastic blood sample in the differential diagnosis versus hyperchromicanemia. c A high proportion of reticulocytes speaks against megaloblasticanemia and for hemolysis (in this case with an absence of pyruvate kinase activity).d Bone marrow in myelodysplasia (type RAEB), with clinical hyperchromicanemia.155d

156Erythrocyte and Thrombocyte AbnormalitiesErythrocyte InclusionsErythrocytes normally show fairly homogeneous hemoglobinization afterpanoptic staining; only after supravital staining (p. 155) will the remainsof their ribosomes show as substantia granulofilamentosa in the reticulocytes.Under certain conditions during the preparation of erythrocytes,aggregates of ribosomal material may be seen as basophilic stippling, alsovisible after Giemsa staining (Fig. 55 a). This is normal in fetal and infantblood; in adults a small degree of basophilic stippling has nonspecific diagnosticvalue, like anisocytosis indicating only a nonspecific reactiveprocess. Not until a large proportion of basophilic stippled erythrocytes isseen concomitantly with anemia does this phenomenon have diagnosticsignificance: thalassemia, lead poisoning, and sideroblastic anemia are allpossibilities. Errant chromosomes from the mitotic spindle are sometimesfound in normoblasts as small spheres with a diameter of about 1 µm,which have remained in the erythrocyte after expulsion of the nucleus andlie about as unevenly distributed Howell–Jolly bodies in the cytoplasm(Fig. 55 b, c). Normally, these cells are quickly sequestered by the spleen.Always after splenectomy, rarely also in hemolytic conditions and megaloblasticanemia, they are observed in erythrocytes at a rate of 1‰.These Howell-Jolly bodies are visible after normal panoptic staining.Supravital staining (see reticulocyte count, p. 11) may produce a fewglobular precipitates at the cell membrane, known as Heinz bodies. Theyare a rare phenomenon and may indicate the presence of unstablehemoglobins in rare familial or severe toxic hemolytic conditions (p. 144).An ellipsoid, eosinophil-violet stained, delicately structured ring inerythrocytes is called a Cabot ring (Fig. 55 d). It probably consists not, as itsform might suggest, of remnants of the nuclear membrane, but of fibersfrom the mitotic spindle. Because these ring structures are sporadicallyseen in all severe anemias, no specific conclusions can be drawn from theirpresence, but in the absence of any other rationale for severe anemia theyare compatible with the suspicion of an incipient idiopathic erythropoieticdisorder (e.g., panmyelopathy or smoldering leukosis orleukemia).

Small inclusions are usually a sign of nonspecific anomalies orartifactsbacdefgFig. 55 Erythrocyte inclusions. a Polychromatic erythrocyte with fine, dense basophilicstippling. b Erythrocyte with Howell–Jolly bodies (arrow) in addition to alymphocyte (after splenectomy). c Erythrocyte with two Howell–Jolly bodies (arrow)alongside an orthochromatic erythroblast with basophilic stippling (thalassemia,in this case with functional asplenia). d Erythrocyte with a delicate Cabotring (arrow) (here in a case of osteomyelosclerosis). e Thrombocyte layered ontoan erythrocyte (arrow). f and g Fixation and staining artifacts.157

158Erythrocyte and Thrombocyte AbnormalitiesHematological Diagnosis of MalariaVarious parasites may be found in the blood stream, e.g., trypanosomesand filariae. Among the parasitic diseases, probably only malaria is ofpractical diagnostic relevance in the northern hemisphere, while at thesame time malarial involvement of erythrocytes may confuse the interpretationof erythrocyte morphology. For these reasons, a knowledge ofthe principal different morphological forms of malarial plasmodia isadvisable.Recurrent fever and influenza-like symptoms after a stay in tropical regionssuggest malaria. The diagnosis may be confirmed from normalblood smears or thick smears; in the latter the erythrocytes have beenhemolyzed and the pathogens exposed. Depending on the stage in the lifecycle of the plasmodia, a variety of morphologically completely differentforms may be found in the erythrocytes, sometimes even next to eachother. The different types of pathogens show subtle specific differencesthat, once the referring physician suspects malaria, are best left to thespecialist in tropical medicine, who will determine which of the followingis the causative organism: Plasmodium vivax (tertian malaria), Plasmodiumfalciparum (falciparum or malignant tertian malaria) and Plasmodiummalariae (quartan malaria) (Table 27, Fig. 56). Most cases ofmalaria are caused by P. vivax (42%) and P. falciparum (43%).The key morphological characteristics in all forms of malaria can besummarized as the following basic forms: The first developmental stage ofthe pathogen, generally found in quite large erythrocytes, appears as smallring-shaped bodies with a central vacuole, called trophozoites (or thesignet-ring stage) (Fig. 57). The point-like center is usually most noticeable,as it is reminiscent of a Howell–Jolly body, and only a very carefulsearch for the delicate ring form will supply the diagnosis. Occasionally,there are several signet-ring entities in one erythrocyte. All invaded cellsmay show reddish stippling, known as malarial stippling or Schüffner’sdots. The pathogens keep dividing, progressively filling the vacuoles, andthen develop into schizonts (Fig. 56), which soon fill the entire erythrocytewith an average of 10–15 nuclei. Some schizonts have brown-black pigmentinclusions. Finally the schizonts disintegrate, the erythrocyte ruptures,and the host runs a fever. The separate parts (merozoites) then start anew invasion of erythrocytes.In parallel with asexual reproduction, merozoites develop into gamonts,which always remain mononuclear and can completely fill theerythrocyte with their stippled cell bodies. The large, dark blue-stainedforms (macrogametes) are female cells (Fig. 57 d), the smaller, light bluestainedforms (microgametes) are male cells. Macrogametes usually predominate.They continue their development in the stomach of the infectedAnopheles mosquito.

PlasmodiumfalciparumPlasmodiumvivaxPlasmodiumovalePlasmodiummalariaeMaturemicrogametocyteMaturemacrogametocyteMatureschizontIntermediatetrophozoiteYoungtrophozoiteFig. 56 Differential diagnosis of malaria plasmodia in a blood smear (from Kayser,F., et al., Medizinische Mikrobiologie (Medical Microbiology), Thieme, Stuttgart,1993).159

160Erythrocyte and Thrombocyte AbnormalitiesThe example shown here is of the erythrocytic phases of P. vivax, becausetertian malaria is the most common form of malaria and best showsthe basic morphological characteristics of a plasmodia infection.Table 27 Parasitology of malaria and clinical characteristics (from Diesfeld, H., G.Krause: Praktische Tropenmedizin und Reisemedizin. Thieme, Stuttgart 1997)Disease Pathogen Exoerythrocyticphase incubationErythrocyticphaseClinical characteristicsFalciparummalariaTertianmalariaQuartanmalariaPlasmodiumfalciparumP. vivaxP. ovale⎫⎪⎬⎪⎭7–15 days, doesnot form liverhypnozoites12-18 days,forms liverhypnozoitesP. malariae 18-40 days,does not formliver hypnozoites48 hours,recurringfever is rare48 hoursPotentially lethaldisease, widelytherapy-resistant,there is usually norecurrence afterrecoveryBenign form recurrencesup to 2 yearsafter infectionBenign form recurrencesup to 5 yearsafter infection72 hours Benign form recurrencespossible upto 30 years afterinfection

Conspicuous erythrocyte inclusions suggest malariaabcdeFig. 57 Blood analysis in malaria. a Trophozoites in Plasmodium falciparum (falciparumor malignant tertian malaria) infection. Simple signet-ring form (1), double-invadederythrocyte with basophilic stippling (2). Erythrocyte with basophilicstippling without plasmodium (3) and segmented neutrophilic granulocyte withtoxic granulation (4). b and c Plasmodium falciparum: single invasion with delicatetrophozoites (1), multiple invasion (2). d and e In the gametocyte stage of falciformmalaria, the pathogens appear to reside outside the erythrocyte, but remnantsof the erythrocyte membrane may be seen (arrow, e). For a systematic overviewof malarial inclusions, see p. 159.161

162Erythrocyte and Thrombocyte AbnormalitiesPolycythemia Vera (ErythremicPolycythemia) and ErythrocytosisIncreases in erythrocytes, hemoglobin, and hematocrit above the normalrange due to causes unrelated to hematopoiesis (i.e., the majority of cases)are referred to as secondary erythrocytosis or secondary polycythemia.However, it should be remembered that the “normal” range of values isquite wide, especially for men, in whom the normal range can be as muchas 55% of the hematocrit!Table 28Causes of secondary erythrocytosisReduced O 2 transport capacityReduced O 2 release from hemoglobinRenal hypoxiaAutonomous erythropoietin biosynthesis– Carboxyhemoglobin formation in chronicsmokers– High altitude hypoxia, COPD– Heart defect (right–left shunt)– Hypoventilation (e.g., obesity hypoventilation)– Congenital 2,3-diphosphoglycerate deficiency– Hydronephrosis, renal cyst– Renal artery stenosis– Renal carcinoma– AdenomaCOPD = Chronic obstructive pulmonary diseaseAn autonomous increase in erythropoiesis, i.e., polycythemia vera, representsa very different situation. Polycythemia vera—which one mightcall a “primary erythrocytosis“—is a malignant stem cell abnormality ofunknown origin and is a myeloproliferative syndrome (p. 112). Leukocytecounts (with increased basophils) and thrombocyte counts often risesimultaneously, and a transition to osteomyelosclerosis often occurs inthe later stages.Revised Criteria for the Diagnosis of Polycythemia Vera (according toPearson and Messinezy, 1996)— A1 elevated erythrocyte numbers— A2 absence of any trigger of secondary polycythemia— A3 palpable splenomegaly— A4 clonality test, e.g., PRV-1 marker (polycythemia rubra vera 1)— B1 thrombocytosis ( 400 10 9 /l)— B2 leukocytosis ( 10 10 9 /l)— B3 splenomegaly (sonogram)— B4 endogenous erythrocytic coloniesDiagnosis in the presence of: A1 + A2 + A3/A4 or A1 + A2 + 2 BBone marrow cytology shows increased erythropoiesis in both polycythemiavera and secondary polycythemia. Polycythemia vera is themore likely diagnosis when megakaryocytes, granulopoiesis, basophils,and eosinophils are also increased.

Bone marrow analysis contributes to the differential diagnosisbetween secondary erythrocytosis and polycythemia veraabcdeFig. 58 Polycythemia vera and secondary erythrocytosis. a In reactive secondaryerythrocytosis there is usually only an increase in erythropoiesis. b In polycythemiavera megakaryopoiesis (and often granulopoiesis) are also increased. c Bonemarrow smear at low magnification in polycythemia vera, with a hyperlobulatedmegakaryocyte (arrow). d Bone marrow smear at low magnification in polycythemiavera, showing increased cell density and proliferation of megakaryocytes. e Inpolycythemia vera, iron staining shows no iron storage particles.163

164Erythrocyte and Thrombocyte AbnormalitiesThrombocyte AbnormalitiesEDTA in blood collection tubes (e.g., lavender top tubes) can lead toaggregation (pseudothrombocytopenia). A control using citrate as anticoagulantis required.The clinical sign of spontaneous bleeding in small areas of the skin andmucous membranes (petechial bleeding), which on injury diffuses out toform medium-sized subcutaneous ecchymoses, is grounds for suspicionof thrombocyte (or vascular) anomalies.ThrombocytopeniaThrombocytopenias Due to Increased Demand (High Turnover)A characteristic sign of this pathology can be that among the few thrombocytesseen in the blood smear, there is an increased presence of larger,i.e., less mature cell forms.The bone marrow in these thrombocytopenias shows raised (or at leastnormal) megakaryocyte counts. Here too, there may be an increased presenceof less mature forms with one or two nuclei (Figs. 60, 61).Drug-induced immunothrombocytopenia As in drug-induced agranulocytosis,the process starts with an antibody response to a drug, or its metabolites,and binding of the antibody complex to thrombocytes. Rapidthrombocyte degradation by macrophages follows (Table 29). The mostcommon triggers are analgesic/anti-inflammatory drugs and antibiotics.

Thrombocytes: increases, reductions, and anomalies may beseenabFig. 59 Forms of thrombocytopenia. a This blood smear shows normal size anddensity of thrombocytes. b In this blood smear thrombocyte density is lower andsize has increased, a feature typical of immunothrombocytopenia. continued 165

166Erythrocyte and Thrombocyte AbnormalitiesTable 29Most common triggers of drug-induced immunothrombocytopeniaAnalgesicsAntibioticsAnticonvulsive drugsArsenic, e. g., in waterQuinidineQuinineDigitalis preparationsFurosemideGold saltsHeparin!IsoniazidMethyldopaSpironolactoneTolbutamide“Idiopathic” immunothrombocytopenia. The name is largely historical,because often a trigger is found.➤ Postinfectious = acute form, usually occurs in children after rubella,mumps, or measles.➤ Essential thrombocytopenia, thrombocytopenic purpura (Werlhof disease)Thrombocyte antibodies develop without an identifiable trigger. This isthus a primary autoimmune disease specifically targeting thrombocytes.Secondary immunothrombocytopenias, e.g., in lupus erythematosus andother forms of immune vasculitis, lymphomas, and tuberculosis.Post-transfusion purpura occurs mostly in women about one week after ablood transfusion, often after earlier transfusions or pregnancy.Thrombocytopenia in microangiopathy. This group includes thrombotic–thrombocytopenic purpura (see p. 144) and disseminated intravascularcoagulation.Thrombocytopenia in hypersplenism of whatever etiology.Fig. 59 Continued. c Pseudothrombocytopenia. The thrombocytes are not lying free and scattered around, but agglutinated together, leading to a reading ofthrombocytopenia from the automated blood analyzer. d Giant thrombocyte (aslarge as an erythrocyte) in thrombocytopenia. Döhle-type bluish inclusion (arrow)in the normally granulated segmented neutrophilic granulocyte: May-Hegglinanomaly.

Thrombocytes: increases, reductions, and anomalies may beseencdeFig. 59 e Large thrombocyte (1) in thrombocytopenia. Thrombocyte-like fragmentsfrom destroyed granulocytes (cytoplasmic fragments) (2), which have thesame structure and staining characteristics as the cytoplasm of band granulocytes(3). Clinical status of sepsis with disseminated intravascular coagulation. In automatedcounters cytoplasmic fragments are included in the thrombocyte fraction.167

168Erythrocyte and Thrombocyte AbnormalitiesThrombocytopenias Due to Reduced Cell ProductionIn this condition, blood contains few, usually small, pyknotic (“old”)thrombocytes. Only a very few megakaryocytes are found in the bonemarrow, and these have a normal appearance.CausesChronic alcoholism. There may be some overlap with increased turnoverand folic acid deficiency.Chemical and radiological noxae. Cytostatics naturally lead directly to adose-dependent reduction in megakaryocyte counts. A large therapeuticradiation burden in the area of blood-producing bone marrow has thesame result, which may persist for many months.Virus infections. Measles, mononucleosis (Epstein–Barr virus, cytomegalovirus),rubella, and influenza may (usually in children) trigger thrombocytopeniasof various types. In these cases, the virus affects the megakaryocytesdirectly. However, antibodies to thrombocytes may also arisein the course of these infections (p. 166), so that the pathomechanism ofthe parainfectious thrombocytopenias described by Werlhof in childrenmay lie in impaired production and/or increased degradation of thrombocytes.Neoplastic and aplastic bone marrow diseases. All neoplasms of the bonemarrow cell series (e.g., leukemia, lymphoma, and plasmacytoma), togetherwith their precursor forms (e.g., myelodysplasia), lead to progressivethrombocytopenia, as do panmyelophthisis and bone marrow infiltrationby metastases from solid tumors.Vitamin deficiency. Folic acid and vitamin B 12 deficiencies from variouscauses (p. 152) also affect the rapidly proliferating megakaryocytes. Inthese cases, thrombocytopenia is often present before anemia andleukocytopenia in circulating blood, while the bone marrow showscopious megakaryocytes that have been blocked from maturation.Constitutional diseases. An amegakaryocytic thrombocytopenia withoutany of the above causes is rare. It is seen in children with congenital radialaplasia; in adults it tends usually to be an early sign of leukemia,myelodysplastic syndrome, or aplastic anemia.Wiscott-Aldrich syndrome (thrombocytopenia, immune deficiency, andeczema) is an X-chromosomal recessive disease in boys and presents withthrombocytopenia with ineffective megakaryopoiesis.The May-Hegglin anomaly (dominant hereditary transmission) ischaracterized by thrombocytopenia with giant thrombocytes andgranulocyte inclusions, which resemble Döhle bodies (endoplasmatic reticulumaggregates).

Variant forms of thrombocyte and megakaryocyte morphologyin the bone marrow are diagnostic aids in thrombocytopeniaabcdefFig. 60 Morphology of thrombocytes and megakaryocytes. a Bone marrow inthrombocytopenia due to increased turnover (e.g., immunothrombocytopenia).Mononuclear “young” megakaryocytes clearly budding a thrombocyte (irregular,cloudy cytoplasm structure). b In thrombocytopenia against a background ofmyelodysplasia, the bone marrow shows various megakaryocyte anomalies: here,too small a nucleus surrounded by too wide cytoplasm. c–f In myelodysplasia(c and d) and acute myeloid leukemia (e and f), bizarre anomalous thrombocyteshapes (arrows) may occasionally be found.169

170Erythrocyte and Thrombocyte AbnormalitiesThrombocytosis (IncludingEssential Thrombocythemia)Reactive. Thrombocytosis in the form of a constant elevation of thrombocytecounts above an upper normal range of 300 000–450 000/µl, may bereactive, i.e., may appear in response to various tumors (particularlybronchial carcinoma), chronic inflammation (particularly ulcerative colitis,primary chronic polyarthritis), bleeding, or iron deficiency. Thepathology of this form is not known.Essential ThrombocythemiaEssential thrombocythemia, by contrast, is a myeloproliferative disease(see p. 114) in which the main feature of increased thrombocytes is accompaniedby other signs of this group of diseases that may vary in severity,such as leukocytosis and an enlarged spleen. Severe thrombocythemiamay also be seen in osteomyelosclerosis, polycythemia vera, and chronicmyeloid leukemia, and for this reason the following specific diagnostic criteriahave been suggested:Diagnostic criteria for essential thrombocytopenia (according toMurphy et al.)➤ Thrombocytes 600 10 9 /l (with control)➤ Normal erythrocyte mass or Hb 18.5 g/dl , 16.5 g/dl ➤ No significant bone marrow fibrosis➤ No splenomegaly➤ No leukoerythroblastic CBC➤ Absence of morphological or cytogenetic criteria of myelodysplasia➤ Secondary thrombocytosis (iron deficiency, inflammation, neoplasia,trauma, etc.) excludedLarge thrombocytes are found in the peripheral blood smear. However,these also occur in polycythemia vera and osteomyelosclerosis.Bone marrow cytology will show markedly elevated megakaryocytecounts, with the cells often forming clusters and often with hypersegmentednuclei.Fig. 61 Essential thrombocythemia. a Increased thrombocyte density and markedanisocytosis in essential thrombocythemia. b Large thrombocytes (1) and amicro(mega)karyocyte nucleus (2) in essential thrombocythemia. Micro(mega)karyocytesare characterized by a small, very dense and often lobed nucleuswith narrow, uneven cytoplasm, the processes of which correspond to thrombocytes(arrow).

Thrombocyte proliferation with large megakaryocytes: essentialthrombocythemia, a chronic myeloproliferative diseaseabcdFig. 61 c and d Bone marrow cytology in essential thrombocythemia: there is astriking abundance of very large, hyperlobulated megakaryocytes (c); such megakaryocytesmay also be seen in polycythemia vera. d Size comparison with basophilicerythroblasts (arrow). The cloudy cytoplasm of the megakaryocyte is typicalof effective thrombocyte production.171

172

Cytology of Organ Biopsiesand Exudates** Special thanks to Dr. T. Binder, Wuppertal,for the generous gift of several preparations.

174Cytology of Organ Biopsies and ExudatesIn this guide to morphology, only a basic indication can be given of thematerials that may be drawn upon for a cytological diagnosis and whatbasic kinds of information cytology is able to give.For specialized cytological organ diagnostics, the reader should refer toa suitable cytology atlas. Often appropriately prepared samples are oftensent away to a hematological–cytological or a pathoanatomic laboratoryfor analysis. Thus, the images in this chapter are intended particularly tohelp the clinician understand the interpretation of samples that he or shehas not investigated in person.In principle, all parenchymatous organs can be accessed for material forcytological analysis. Of particular importance are thyroid biopsy (especiallyin the region of scintigraphically “cold” nodules), liver and spleenbiopsy (under laparascopic guidance) in the region of lumps lying close tothe surface, and breast and prostate biopsy. Again, the cytological analysisis usually made by a specialist cytologist or pathologist.Lymph node cytology, effusion cytology (pleura, ascites), cerebrospinalfluid cytology, and bronchial lavage are usually the responsibility of theinternist with a special interest in morphology and are closely related tohemato-oncology.Lymph Node CytologyThe diagnosis of enlarged lymph nodes receives special attention here becauselymph nodes are as important as bone marrow for hematopoiesis.While in most instances abnormalities in the bone marrow cell series canbe detected from the peripheral blood, this is very rarely the case for lymphomas.For this reason, lymph node cytology, a relatively simple andwell-tolerated technique (p. 24), is critically important for the guidance itcan give about the cause of enlarged lymph nodes. Figure 62 offers a diagnosticflow chart.

Lymph Node Cytology175Anamnesis– sudden fast swelling– possible onset in youth– possible contactwith animals– possible feverslow onset, unclearsymptoms: subfebrile,night sweat, weightlossFindings– pressure pain– focal, or distributedover severallocationsDindolent often in onelocation, possiblylocalization ofprimary tumorBloodanalysis– relative lymphocytosiswithstimulated formsspecific cell presentation(e.g. CLL, immunocytoma,ALL and others)Serology/ – mononucleosis testimmunology – toxoplasmosis– rubella– syphilis– tuberculin skin testDor unspecific signsor Searchfor diseasefocus– e.g. teeth– sinuses– infections ofthe genitalia– if findings arenegative or unsuccessfultherapyafter 1 weekconsidered reactive ifthe lymph node swellingdoes not recede after2 weeks or the causeis foundDLymph nodebiopsysuspicion of tumor ormalignant lymphomaattempt to clarifythe diagnosis after histologypossiblyDdiagnosis basedon histologyDiagnostic flow chart for cases of lymph node enlargement. (D) Diagno-Fig. 62sis.

176Cytology of Organ Biopsies and ExudatesReactive Lymph Node Hyperplasia andLymphogranulomatosis (Hodgkin Disease)Reactive lymph node hyperplasia of whatever etiology is characterized by aconfused mixture of small, middle-sized, and large lymphocytes. Whenthe latter have a nuclear diameter at least three times the size of the predominatingsmall lymphocytes and have a fair width of basophilic cytoplasm,they are called immunoblasts (lymphoblasts). Cells with deeplybasophilic, eccentric cytoplasm and dense nuclei are called plasmablasts,and cells with a narrow cytoplasmic seam are centroblasts. Lymphocytescan also to varying degrees show a tendency to appear as plasma cells, e.g.,as plasmacytoid lymphocytes with a relatively wide seam of cytoplasm.Monocytes and phagocytic macrophages are also seen (Fig. 63).Table 30 (p. 178) summarizes the different forms of reactive lymphadenitis.The basic cytological findings in all of them is always a completemixture of small to very large lymphocytes. Occasionally more specificfindings may indicate the possibility of mononucleosis (increased immaturemonocytes) or toxoplasmosis (plasmablasts, phagocytic macrophages,and possibly epithelioid cells).Any enlargement of the lymph nodes that persists for more than twoweeks should be subjected to histological analysis unless the history, clinicalfindings, serology, or CBC offer an explanation.At first sight, the confusion visible in the cytological findings of lymphogranulomatosis(Hodgkin disease) is reminiscent of the picture in reactivehyperplasia (something which may be important for an understanding ofthe pathology of this disease compared with other malignant neoplasms).However, some cells elements show signs of a strong immunological“over-reaction” in which large, immunoblast-like cells form with welldevelopednucleoli (Hodgkin cells). Sporadically, some of these cells arefound to be multinucleated (Reed–Sternberg giant cells); infiltrations ofeosinophils and plasma cells may also be found. Findings of this type alwaysrequire histological analysis, which can distinguish between fourprognostically relevant histological subtypes. In addition to this, the verylack of a clear demarcation between Hodgkin disease and reactive conditionsis reason enough to conduct a histological study of every lymph nodethat appears reactive if does not regress completely within two weeks.In cases of histologically verified Hodgkin disease, cytological analysisis especially useful in the assessment of new lymphomas after therapy.

Reactive lymph node hyperplasia and lymphogranulomatosis(Hodgkin disease): a polymorphous mixture of cellsabcFig. 63 Reactive lymph node hyperplasia and lymphogranulomatosis. a Lymphnode cytology in severe reactive hyperplasia. Large blastic cells alongside smalllymphocytes (if it fails to regress, histological analysis is required). b Hodgkindisease: a giant mononuclear cell with a large nucleolus (arrow) and wide cytoplasmiclayer (Hodgkin cell), surrounded by small and medium-sized lymphocytes.c Hodgkin disease: giant binuclear cell (Reed–Sternberg giant cell).177

178Cytology of Organ Biopsies and ExudatesTable 30 Sequence of steps in the diagnosis of reactive lymphomaAnamnesis Symptoms Tentative diagnosis Diagnostic studies Cytology HistologyRapid progression(fever)Localized, painful Perifocal lymphadenitisSearch for diseasefocus, non-specificchanges in CBC andESR() Non-specificlymphadenitisRapid progression,fever,sore throatDiffuse, painfult;angina, possiblyspleenMononucleosis Pfeiffer cells in theblood analysis, EBVserology (1)() Adenitis withhistiocytosisMost children Nuchal lymphnodes, laterexanthemaRubella Plasma cells in theblood,rubella-AHT (2)Ingestion of rawmeatContact with catsDiffuse Toxoplasmosis Serology (3) () Withepithelioid cells andmacrophagesEpithelioid celllymphadenitisPharyngitis( conjunctivitis)LocalNeck areaCommon viruses,specific adenovirusesComplement fixationreaction (adenoviruses,less commonlycoxsackieviruses)(4)Slow growth,general malaiseInflamed lymphnodes, possiblyfistulaeTuberculosis Thorax, local primaryinfections, skin test,pathogen in biopsyaspirate, possibly PCR() Epithelioidcells, Langhansgiant cellsSlow growth Hard; possibly skininfiltrationsSarcoidosis (Boeckdisease)Thorax, tuberculintest usually negative,ACE Epithelioid cells Epithelioid cellfibrosis

Lymph Node Cytology179Contact withanimalsTonsillitis, necklymph nodesListeriosis Agglutination test,complement fixationreaction() It may be possibleto determine thepathogen from thebiopsyContact withanimalsMilk intakeFever, spleen Brucellosis In case of fever:pathogen in blood,serologyOpen wound,possibly from acatLocal primary lesion Cat-scratch diseaseLeukocytosis, lymphocytosis,complementfixation reaction Epithelioid cells,giant cellsPerforatinglymphadenitisContact withwildlifeLocal primary lesion Tularemia (rabbitfever)Agglutination testLittle sense ofillnessInflamed hard infiltrates,possiblyfistulaeActinomycosis Leukocytosis, left shift “Gland” tissue inthe biopsy materialTherapeuticexcisionSymptomaticjointsJoints, spleen,possibly kidneyCollagen disease(PCP, LE) Felty syndrome,Still diseaseAntinuclear factorNo symptoms Submandibularswelling, no irritationBranchial cyst Epithelial cells,macrophages, andgranulocytesTherapeuticexcision() = Optional step; = usually diagnostic step, if there is no arrow, the diagnosis can be made on the basis of preceding steps. (1) = Positive from day 5;(2) = 1–4 days after exanthema, may be as much as 1 month; (3) = from week 4; (4) = from day 10.

180Cytology of Organ Biopsies and ExudatesSarcoidosis and TuberculosisThe material of cell biopsies taken from indolent, nonirritated enlargedlymph nodes in the neck or axilla that have developed with little in theway of clinical symptoms, or from subcutaneous infiltration in various regions,can be quite homogeneous. With their thin, very long, ovoid nucleus(four to five times the size of lymphocytes), delicate reticular chromatinstructure, and extensive layer of cytoplasm that may occasionallyappear confluent with that of other cells, they are reminiscent of theepithelial cells that line the body’s internal cavities and are thereforecalled epithelioid cells. They are known to be the tissue form of transformedmonocytes, and are found in increased numbers in all chronic inflammatoryprocesses—especially toxoplasmosis, autoimmune diseases,and foreign-body reactions—and also in the neighborhood and drainageareas of tumors. They exclusively dominate the cytological picture in aparticular form of chronic “inflammation,” sarcoidosis (Boeck disease). Atypical finding almost always encountered at the pulmonary hilus combinedwith a negative tuberculin test will all but confirm this diagnosis.The appearance of a few multinuclear cells (Langhans giant cells) mayallow confusion with tuberculosis, but clinical findings and a tuberculinskin test will usually make the diagnosis clear.Rapidly developing, usually hard, pressure-sensitive neck lymph nodes,seemingly connected with each other with some fluctuant zones and externalinflammatory redness, suggest the now rare scrofulous form oftuberculosis. A highly positive tuberculin skin test also suggests this diagnosis.If any remaining doubts cannot be dispelled clinically, a very-fineneedlelymph node biopsy may be performed, but only if the skin showsnoninflammatory, pale discoloration.The harvested material can show the potency of the tissue-boundforms of cells in the monocyte/macrophage series. In addition to mononuclearepithelioid cells, there are giant cell conglomerates made up ofpolynuclear epithelioid cells in enormous syncytia with 10, 20, or morenuclei. These are called Langhans giant cells. In scrofuloderma (tuberculosiscolliquativa), there are also lymphocytic and granulocytic cells in theprocess of degradation, which are absent in purely productive tuberculouslymphadenitis.

Epithelioid cells dominate the lymph node biopsy: Boeck diseaseor tuberculosisabFig. 64 Boeck disease and tuberculosis. a Lymph node cytology in Boeck disease:a special form of reactive cell pattern with (often predominating) islandsand trains of epithelioid cells (arrow), which have ovoid nuclei with delicate chromatinstructure and a wide, smoke-gray layer of cytoplasm. b Lymph node cytologyin tuberculous lymphadenitis: in addition to lymphocytes and a few epithelialcells (1), enormous syncytes of epithelioid cell nuclei within one cytoplasm (arrow)may be encountered: the Langhans giant cell.181

182Cytology of Organ Biopsies and ExudatesNon-Hodgkin LymphomaSince the CBC is the first step in any lymph node diagnosis, lymph node biopsyis unnecessary in many cases of non-Hodgkin lymphoma (p. 70), becausethe most common form of this group of diseases, chronic lymphadenosis,can always be diagnosed on the basis of the leukemic findings of theCBC.However, when enlarged lymph nodes are found in one or more regionswithout symptoms of reactive disease, and the blood analysis fails to showsigns of leukemia, lymph node biopsy is indicated.The relatively monotonous lymph node cytology in non-Hodgkin lymphomasand tumor metastases mean that histological differentiation isrequired.In contrast to Hodgkin disease, with its conspicuous giant cell forms(p. 177), non-Hodgkin lymphomas display a monotonous picture withoutany signs of a reactive process (p. 70). Clinically, it is enough to distinguishbetween small cell forms (which have a relatively good prognosis) andlarge cell forms (which have a poorer prognosis) to begin with. For a moredetailed classification, see page 70 f.Histological analysis may be omitted only when its final results wouldnot be expected to add to the intermediate cytological findings in terms ofconsequences for treatment.Metastases of Solid Tumors in Lymph Nodes orSubcutaneous TissueWhen hard nodules are found that are circumscribed in location, biopsyshows aggregates of polymorphous cells with mostly undifferentiated nucleiand a coarse reticular structure of the chromatin (perhaps with welldefinednucleoli or nuclear vacuoles), and the lymphatic cells cannot beclassified, there is urgent suspicion of metastasis from a malignant solidtumor, i.e. from a carcinoma in a variety of possible locations or a softtissue sarcoma.As a rule, the next step is the search for a possible primary tumor. If this isfound, lymph node resection becomes unnecessary.If no primary tumor is found, lymph node histology is indicated. The histologicalfindings can provide certain clues about the etiology and also helpsin the difficult differential diagnosis versus blastic non-Hodgkin lymphoma.

In cases of non-Hodgkin lymphoma and tumor metastases, atentative diagnosis is possible on the basis of the lymph nodecytologyabcdeFig. 65 Non-Hodgkin lymphoma and tumor metastases. a Lymph node cytologyshowing small cells with relatively wide cytoplasm (arrow 1) in addition to lymphocytes.There are scattered blasts with wide cytoplasm (arrow 2): lymphoplasmacyticimmunocytoma. b Lymph node cytology showing exclusively large blastoidcells with a large central nucleolus (arrow). This usually indicates large-cell non-Hodgkin lymphoma (in this case immunoblastic). c–e Metastatic disease from:c uterine carcinoma, d small-cell bronchial carcinoma, and e leiomyosarcoma.183

184Cytology of Organ Biopsies and ExudatesBranchial Cysts and BronchoalveolarLavageBranchial CystsA (usually unilateral) swollen neck nodule below the mandibular anglethat feels firm to pressure, but is without external signs of inflammation,should suggest the presence of a branchial cyst. Surprisingly, aspirationusually produces a brownish-yellow liquid. In addition to partially cytolysedgranulocytes and lymphocytes (cell detritus), a smear of this liquid,or the centrifuged precipitate, shows cells with small central nuclei andwide light cell centers which are identical to epithelial cells from the floorof the mouth. Biopsies from a soft swelling around the larynx show thesame picture; in this case it is a retention cyst from another developmentalremnant, the ductus thyroglossus.Cytology of the Respiratory System,Especially Bronchoalveolar LavageThrough the development of patient-friendly endoscopic techniques, diagnosticlavage (with 10–30 ml physiological saline solution) and its cytologicalworkup are now in widespread use. This method is briefly mentionedhere because of its broad interest for all medical professionals withan interest in morphology; the interested reader is referred to thespecialist literature (e.g. Costabel, 1994) for further information. Table 31lists the most important indications for bronchoalveolar lavage.Table 311994)Clinical indications for bronchoalveolar lavage (according to CostabelInterstitial infiltrates Alveolar infiltrates Pulmonary infiltrates inpatients with immunedeficiencySarcoidosis (Boeck disease)Exogenous allergic alveolitisDrug-induced alveolitisIdiopathic pulmonary fibrosisCollagen diseaseHistiocytosis XPneumoconiosesLymphangiosis carcinomatosaPneumoniaAlveolar hemorrhageAlveolar proteinosisEosinophilic pneumoniaObliterating bronchiolitisHIV InfectionTreatment with cytostaticagentsRadiation sicknessImmunosuppressive therapyOrgan transplant

Accessible cysts (e.g., branchial cysts) should be aspirated. Bronchiallavage is a cytological new disciplineabcdFig. 66 Cyst biopsy and bronchoalveolar lavage. a Cytology of a lateral neck cyst:no lymphatic tissue, but epithelial cells from the floor of the mouth. b Normalciliated epithelial cells with typical cytoplasmic processes. c Tumor cell conglomerationin small-cell bronchial carcinoma: conglomeration is typical of tumor cells.d Bronchoalveolar lavage in purulent bronchitis: a macrophage with pigmentinclusion (arrow) is surrounded by segmented neutrophilic granulocytes.185

186Cytology of Organ Biopsies and ExudatesCytology of Pleural Effusions and AscitesPleural effusions always require cytological diagnostic procedures unlessthey are secondary to a known disease, such as cardiac insufficiency orpneumonia, and recede on treatment of the primary disease.Pleura aspirates can be classified as exudates or transudates (the latterusually caused by hydrodynamic stasis). The specific density (measuredwith a simple areometer) of transudates, which are protein-poor, is between1008 and 1015 g/l, while for exudates it is greater than 1018 g/l.Cytological preparation may be done by gentle centrifugation of theaspirate (10 minutes at 300–500 rpm), which should be as fresh aspossible; the supernatant is decanted and the sediment suspended in theresidual fluid, which will collect on the bottom of the centrifuge tube.Nowadays, however, this procedure has been replaced by cytocentrifugation.Effusions that are noticeably rich in eosinophilic granulocytes shouldraise the suspicion of Hodgkin disease, generalized reaction to the presenceof a tumor, or an allergic or autoimmune disorder. Purely lymphaticeffusions are particularly suggestive of tuberculosis. In addition, all transudatesand exudates contain various numbers of endothelial cells (particularlyhigh in cases of bacterial pleuritis) that have been sloughed off fromthe pleural lining.Any cell elements that do not fulfill the above criteria should be regardedas suspect for neoplastic transformation, especially if they occur inaggregates. Characteristics that in general terms support such a suspicioninclude extended size polymorphy, coarse chromatin structure, welldefinednucleoli, occasional polynucleated cells, nuclear and plasmavacuoles, and deep cytoplasmic basophilia. For practical reasons, specialdiagnostic procedures should always be initiated in these situations.What was said above in relation to the cell composition of pleural effusionsalso holds for ascites. Here too, the specific density may be determinedand the Rivalta test to distinguish exudate from transudate carriedout. Inflammatory exudates usually have a higher cell content; a strongpredominance of lymphocytes may indicate tuberculosis. Like the pleura,the peritoneum is lined by phagocytotic endothelial cells which slough offinto the ascitic fluid and, depending on the extent of the fluid, may producea polymorphous overall picture analogous to that of the pleural endothelialcells. It is not always easy to distinguish between such endothelialcells and malignant tumor metastases. However, the latter usuallyoccur not alone but in coherent cell aggregates (“floatingmetastases”), the various individual elements of which typically show acoarse chromatin structure, wide variation in size, well-defined nucleoli,and deeply basophilic cytoplasm.

Tumor cells can be identified in pleural and ascites smearsabcdFig. 67 Pleural effusion and ascites. a Pleural cytology, nonspecific exudate: dormantmesothelial cell (or serosal cover cell) (1), phagocytic macrophage with vacuoles(2), and monocytes (3), in addition to segmented neutrophilic granulocytes(4). b Cell composition in a pleural aspirate (prepared using a cytocentrifuge): variablecells, whose similarity to cells in acute leukemia should be established by cytochemistryand marker analysis: lymphoblastic lymphoma. c Ascites with tumorcell conglomerate, surrounded with granulocytes and monocytes, in this case ofovarian carcinoma. d Ascites cytology with an island of tumor cells. This kind ofconglomeration is typical of tumor cells.187

188Cytology of Organ Biopsies and ExudateCytology of Cerebrospinal FluidThe first step in all hemato-oncological and neurological diagnosticassessments of cerebrospinal fluid is the quantitative and qualitativeanalysis of the cell composition (Table 32).Table 32 Emergency diagnostics of the liquor (according to Felgenhauer in Thomas 1998)➤ Pandy’s reaction➤ Cell count (Fuchs-Rosenthal chamber)➤ Smear (or cytocentrifuge preparation)– to analyze the cell differentiation and– to search for bacteria and roughly determine their types and prevalence➤ Gram stain➤ An additional determination of bacterial antigens may be doneUsing advanced cell diagnostic methods, lymphocyte subpopulationscan be identified by immunocytology and marker analysis and cytogenetictests carried out on tumor cells.Prevalence of neutrophilic granulocytes with strong pleocytosis suggestsbacterial meningitis; often the bacteria can be directly characterized.Prevalence of lymphatic cells with moderate pleocytosis suggests viralmeningitis. (If clinical and serological findings leave doubts, the differentialdiagnosis must rule out lymphoma using immunocytologicalmethods.)Strong eosinophilia suggests parasite infection (e.g., cysticercosis).A complete mixture of cells with granulocytes, lymphocytes, and monocytesin equal proportion is found in tuberculous meningitis.Variable blasts, usually with significant pleocytosis, predominate inleukemic or lymphomatous meningitis.Undefinable cells with large nuclei suggest tumor cells in general, e.g.,meningeal involvement in breast cancer or bronchial carcinoma, etc. Thecell types are determined on the basis of knowledge of the primary tumorand/or by marker analysis. Among primary brain tumors, the most likelycells to be found in cerebrospinal fluid are those from ependymoma,pinealoma, and medulloblastoma.Erythrophages and siderophages (siderophores) are monocytes/macrophages,which take up erythrocytes and iron-containing pigment duringsubarachnoid hemorrhage.The cytological analysis of the cerebrospinal fluid offers important cluesto the character of meningeal inflammation, the presence of a malignancy,or hemorrhage.

Viral, bacterial, and malignant meningitis can be distinguishedby means of cerebrospinal fluid cytologyabcdefghFig. 68 Cerebrospinal fluid cytology. a Cerebrospinal fluid cytology in bacterialmeningitis: granulocytes with phagocytosed diplococci (in this case, pneumococci,arrow). b Cerebrospinal fluid cytology in viral meningitis: variable lymphoidcells. c Cerebrospinal fluid cytology in non-Hodgkin lymphoma: here, mantle celllymphoma. d Cerebrospinal fluid cytology after subarachnoid hemorrhage: macrophageswith phagocytosed erythrocytes. e–h Cerebrospinal fluid cytology inmeningeal involvement in malignancy: the origin of the cells cannot be deducedwith certainty from the spinal fluid cytology alone: (e) breast cancer, (f) bronchialcarcinoma, (g) medulloblastoma, and (h) acute leukemia.189

190ReferencesBegemann, H., M. Begemann: Praktische Hämatologie, 10. Aufl. Thieme, Stuttgart1998Begemann, H., J. Rastetter: Klinische Hämatologie, 4. Aufl. Thieme, Stuttgart 1993Bessis, M.: Blood Smears Reinterpreted. Springer, Berlin 1977Binet, J.L., A. Auquier, G. Dighiero et al.: A new prognostic classification of chroniclymphocytic leukemia derived from a multivariate survival analysis. Cancer1981; 48(1): 198-206Brücher, H.: Knochenmarkzytologie. Thieme, Stuttgart 1986Classen, M., A. Dierkesmann, H. Heimpel et al.: Rationelle Diagnostik und Therapiein der inneren Medizin. Urben & Schwarzenberg, München 1996Costabel, M.: Atlas der bronchoalveolären Lavage. Thieme, Stuttgart 1994Costabel V., J. Guzman: Bronchoalveolar lavage in interstital lung disease. CurrOpin Pulm Med 2001; 7(5): 255-61Durie, B.G.M., S.E. Salmon: A clinical staging system for multiple myeloma. Correlationof measured myeloma cell mass with presenting clinical features, responseto treatment, and survival. Cancer 1975; 36: 842-854Heckner, F., M. Freund: Praktikum der mikroskopischen Hämatologie. Urban &Schwarzenberg, München 1994Heimpel, H., D. Hoelzer, E. Kleihauer, H. P. Lohrmann: Hämatologie in der Praxis.Fischer, Stuttgart 1996Huber, H., H. Löffler, V. Faber: Methoden der diagnostischen Hämatologie.Springer, Berlin 1994Jaffe, E. S., N. L. Harris, H. Stein, J. W. Vardiman: World Health Organization Classificationof Tumours. Pathology and Genetics of Tumours of Haematopoietic andLymphoid Tissues. IARC Press, Lyon 2001Lennert, K., A. C. Feller: Non-Hodgkin-Lymphome. Springer, Berlin 1990Löffler, H., J. Rastetter: Atlas der klinischen Hämatologie. Springer, Berlin 1999Murphy, S.: Diagnostic criteria and prognosis in polycythemia vera and essentialthombocythemia. Semin Hematol 1999; 36(1 Suppl 2): 9-13Ovell, S. R., G. Sterrett, M. N. Walters, D. Whitaker: Fine Needle Aspiration Cytology.Churchill Livingstone, Edinburgh London 1992Pearson, T.C., M Messinezy: The diagnostic criteria of polycythaemia rubra vera.Leuk Lymphoma 1996;22(Suppl 1): 87-93Pralle, H. B.: Checkliste Hämatologie, 2. Aufl. Thieme, Stuttgart 1991Schmoll, H. J., K. Höffken, K. Possinger: Kompendium internistische Onkologie,3. Bde., 3. Aufl. Springer, Berlin 1999Theml, H., W. Kaboth, H. Begemann: Blutkrankheiten. In Kühn, H. A., J. Schirmeister:Innere Medizin, 5. Aufl. Springer, Berlin 1989Theml, H., H. D. Schick: Praktische Differentialdiagnostik hämatologischer undonkologischer Krankheiten. Thieme, Stuttgart 1998Zucker-Franklin, D., M. F. Greaves, C. E. Grossi, A. M. Marmont: Atlas der Blutzellen,2. Aufl. Fischer, Stuttgart 1990

191IndexAActinomycosis 179Addison disease 66Agammaglobulinemia 48Agranulocytosis 48, 86acute 86allergic 8bone marrow analysis 54, 86, 87classification 86subacute 86Alcoholism, chronic 168Allergic processes 8, 44basophilia and 124eosinophilia and 124Anemia 80, 106–107aplastic 8, 48, 56, 131, 140, 148–150bone marrow analysis 53, 54, 56classification 128congenital dyserythropoietic (CDA)147, 148diagnosis 130–131Diamond–Blackfan 146dimorphic 152hemolytic 131, 140–145causes 142microangiopathic 144, 150with erythrocyte anomalies144–145hemorrhage and 131hyperchromic 128, 152–155causes 152hyper-regenerative 128hypochromic 128–139bone marrow analysis 134–137infectious/toxic 131, 134–135, 136iron-deficiency 128–133, 136sideroachrestic 137with hemolysis 138–139hypoplastic 140congenital 146hyporegenerative 128immunohemolytic 8macrocytic 152–153megaloblastic 43, 54, 56, 131,152–154normochromic 128, 140–151bone marrow analysis 140hemolytic 140–145renal 146paraneoplastic 131pernicious 140, 154refractory 106in transformation 108with excess blasts (RAEB) 54, 106with ring sideroblasts 106renal 146secondary 131, 134, 135, 136sickle cell 142, 144, 145sideroachrestic 131, 137sideroblastic 137smoldering 54Anisocytosis 130, 134–135, 137–139,152–153, 170Anulocytes 132, 133Ascites 186–187Auer bodies 89, 96–99Autoantibodies 142Autoimmune processes 8, 180anemia and 134autoagglutination 142, 143eosinophilia and 124Automated blood count 19–20Azurophilic granules 96, 100BB-lymphocytes 48, 49function 6see also LymphocytesBand cells 4, 6, 38–39, 57, 153diagnostic implications 38, 112, 113normal ranges and mean values 12Basophilia 124–126Basophilic stippling 134, 135,137–139, 156–157Basophils 44–45, 125

192IndexBasophils, diagnostic implications 44elevated counts 124–126function 5, 6in chronic myeloid leukemia 118,120, 121normal ranges and mean values 13Bicytopenia 106, 149Blast crisisin chronic myeloid leukemia120–121in osteomyelosclerosis 123Blood cell series 2–5, 53regulation and dysregulation 7–8see also Erythropoiesis; GranulocytopoiesisBlood countsautomated blood counts 19–20complete blood count (CBC) 25differential diagnosis 62–65differential blood count (DBC)17–19, 25–26indications 27erythrocytes 10, 13, 25leukocytes 14, 16, 25reticulocytes 11, 13, 32thrombocytes 15, 25Blood sample collection 9Blood smear 17–19staining 18, 19Blood–bone marrow barrier, destructionof 30, 32, 34Boeck disease 178, 180, 181Bone marrow 52–59analysis 52–59agranulocytosis 54, 86, 87cell density 52chromic myeloid leukemia118–119, 120, 121essential thrombocythemia170–171hypochromic anemias 134–137indications for 26, 27myelodysplasias 108, 109normochromic anemias 140polycythemia vera 162–163red cell series/white cell seriesratios 53, 136space-occupying processes150–151thrombocytopenia 164, 169aplasia 146–150pure red cell aplasia (PRCA)146–148biopsy 20–22, 27cytology 20, 52–55disturbances 8histology 20, 26hyperplasia 149infectious/toxic 134medullary stroma cells 58–59neoplasms 150, 168carcinosis 131, 150–151Bone metastases 150, 168Branchial cyst 179, 184, 185Bronchoalveolar lavage 184–185indications 184Brucellosis 66, 179Bruton disease 48Burkitt lymphoma 71Burr cell 135CCabot ring 156, 157Carcinoma 188, 189bone marrow 131, 150–151metastatic disease 182, 183Cat-scratch disease 179Centroblasts 176Cerebrospinal fluid 188–189Charcot-Leyden crystals 44Chickenpox 66Chromatin structure 4Collagen disease 179Complete blood count (CBC) 25differential diagnosis 62–65Cyclic Murchison syndrome 40Cystbranchial 179, 184, 185retention 184Cytomegalovirus (CMV) infection 66,67, 68, 168DDiagnostic workup 25–27Diamond–Blackfan anemia 146Differential blood count (DBC) 17–19,25–26indications 27DiGeorge disease 48

Index193Disseminated intravascularcoagulopathy (DIC) 144, 167Döhle bodies 39–41Drumstick appendages 42, 43Ductus thyroglossus 184Dyserythropoiesis 102, 106, 109congenital dyserythropoietic anemia(CDA) 147, 148Dysgranulopoiesis 102, 106Dysmegakaryopoiesis 102, 106, 109EElliptocytosis 142Eosinophilia 111, 124, 125cerebrospinal fluid 188Eosinophils 44–45, 125diagnostic implications 44, 56elevated counts 124, 125functions 5, 6in chronic myeloid leukemia 118normal ranges and mean values 12Epstein–Barr virus (EBV) infection 66,68–69, 168Erythremia 54, 162–163Erythroblastopenia 146–148Erythroblastophthisis 146Erythroblastosis 8Erythroblasts 30–33, 57acute erythroleukemia 100basophilic 30, 32, 54, 57, 85diagnostic implications 30, 32orthochromatic 31, 32, 54, 57polychromatic 32–33, 35, 54, 57Erythrocytes 7, 33assessment 26basophilic stippling 134, 135,137–139, 156–157counts 10, 13, 25, 128function 7inclusions 156–161iron deficiency and 132–133normal ranges and mean values 13parameters 10–11red cell distribution width (RDW)11ring-shaped 130, 132, 133tear-drop 115, 123see also AnemiaErythrocytosisprimary 162secondary 162, 163Erythroleukemia 30, 93, 100in normochromic anemia 140Erythrophages 188Erythropoiesis 3, 30–33, 55bone marrow analysis 54, 136iron staining 56, 57, 109dyserythropoiesis 102, 106, 109in hypochromic anemia 136, 138in normochromic anemia 140megaloblastic 54Erythropoietin 146Essential thrombocythemia (ET) 8, 26,56, 114, 170–171diagnosis 170FFaggot cells 99Felty syndrome 179Ferritin granules 56Fibroblastic reticular cells 58, 59Folic acid deficiency 7, 38, 54, 149in hemolytic anemia 140, 141in hyperchromic anemia 152–154thrombocytopenia and 168Fragmentocytes 142, 143Fragmentocytosis 144GGaisböck syndrome 53Gaucher syndrome 118Granulocytes 3, 4, 12, 13degrading forms 42functions 5, 6hypersegmented 153–154precursors 34–37see also Basophils; Eosinophils;NeutrophilsGranulocytopenia 66, 86Granulocytopoiesis 3, 5, 34–39bone marrow analysis 54dysgranulopoiesis 102, 106in acute myelomonocytic leukemia98in hypochromic anemia 136Granulocytosis 110Gumprecht's nuclear shadow 74

194IndexHHeinz bodies 142, 156HELLP syndrome 144Hematocrit 10Hematopoiesisextramedullary 30, 32see also ErythropoiesisHemoglobinassay 10, 128deficiency 132mean cell hemoglobin content(MCH) 10, 13, 128mean cellular hemoglobin concentration(MCHC) 10, 11, 13normal ranges and mean values 13see also AnemiaHemoglobinopathies 144Hemolysis 32, 54, 138–139, 142–143Hemolytic uremic syndrome (HUS)144Hemophagocytosis 46HEMPAS antigen 148Hepatitis 66Histiocytes 118reticular 58sea-blue 118Hodgkin cells 176, 177Hodgkin disease 26, 70, 176, 177eosinophilia 124Howell–Jolly bodies 139, 156, 157Hypereosinophilia syndrome 124Hypergammaglobulinemia 82, 131differential diagnosis 82Hypersensitivity reactions 44eosinophilia and 124Hyperthyroidism 66Hypogammaglobulinemia 74, 82IIgM paraprotein 78Immunoblasts 176Immunocytoma, lymphoplasmacytoid70–71, 74, 77–78, 183Immunothrombocytopenia 165drug-induced 164–166idiopathic 166secondary 166Infection 36, 40, 46, 48, 112–113anemia and 134basophilia and 124eosinophilia and 124thrombocytopenia and 168see also specific infectionsInfectious lymphocytosis 66Infectious mononucleosis 68–69Influenza 168Irondeficiency 7, 56, 128–133blood cell analysis 132bone marrow analysis 136normal ranges 132Iron staining 56, 57, 109Isoantibodies 142LLanghans giant cells 180, 181Leukemia 8, 34, 40, 90–91acute 90–105basophilic 126bone marrow analysis 54characteristics 96classifications 91–93, 94, 104diagnosis 91–94eosinophilic 124erythroleukemia 30, 93, 100lymphocytic (ALL) 104–105classification 104mast cell 126megakaryoblastic 102megakaryocytic 93monoblastic 93, 100–101monocytic 46, 89, 93, 100myeloblastic 96myeloid (AML) 89, 92, 94–97hypoplastic 102, 103with dysplasia 102, 103myelomonocytic 92, 98, 99promyelocytic 92, 98, 99aleukemic 149B-prolymphocytic (B-PLL) 70, 74, 77chronicbasophilic 126lymphocytic (CLL) 66, 70, 74–79,90characteristics 76staging 76, 78myeloid (CML) 26, 44, 56, 90,114–121

Index195blast crisis 120–121bone marrow analysis 118–119,120, 121characteristics 116, 124diagnosis 54, 111, 116–119myelomonocytic (CMML) 89, 90,107, 108eosinophilic 124erythroleukemia 30hairy cell (HCL) 71, 80, 81variant (HCL-V) 80lymphoplasmacytic 71megalokaryocytes 50peroxidase-negative 91peroxidase-positive 91plasma cell 48, 78, 82pseudo-Pelger granulocytes 42T-prolymphocytic 74, 77Leukemic hiatus 91Leukocyte alkaline phosphatase 54Leukocytes 3–4counts 14, 16, 25, 90–91normal ranges and mean values 12Leukocytopenia 80, 107Leukocytosis 63, 110smoker's 111Leukopenia 63Listeriosis 179Loeffler syndrome 124Lung aspirates 186–187Lymph node biopsy 23, 24, 174–183metastatic disease 182, 183non-Hodgkin lymphoma 182, 183reactive lymph node hyperplasia176–179sarcoidosis 180–181tuberculosis 180–181Lymphadenitisreactive 176, 178–179tuberculous 181Lymphadenoma 54, 149chronic 74marginal zone 78, 80Lymphatic cells 63, 66cerebrospinal fluid 188differentiation in non-Hodgkinlymphoma 72–73reactive lymphocytosis 66–67see also LymphocytesLymphoblasts 32, 176acute lymphoblastic leukemia (ALL)104–105Lymphocytes 3, 4, 33, 48–49bone marrow 56diagnostic implications 48, 56, 66functions 6in infectious mononucleosis 68–69large granulated (LGL) 69normal ranges and mean values 12plasmacytoid 77, 176see also Lymphocytosis; LymphomaLymphocytopoiesis 3Lymphocytosis 66–69, 74, 76constitutional relative 66infectious 66reactive 66–67, 86–87Lymphogranulomatosis see HodgkindiseaseLymphoma 69, 77, 82, 174B-cell 70, 71Burkitt 71centroblastic 71centrocytic 71classification 70–72follicular 71, 78, 79large-cell 71, 74lymphoblastic 72lymphoplasmacytic 78malignant 26mantle cell 71, 78, 79, 189marginal zone 71monocytoid 71non-Hodgkin (NHL) 26, 70–73blastic 70cell surface markers 72–73lymph node biopsy 182, 183precursor 70reactive 176–179diagnosis 178–179splenic lymphoma with villouslymphocytes (SLVL) 80, 81T-cell 72cutaneous (CTCL) 74, 77Lymphomatous toxoplasmosis 66MMacroblasts 30Macrocytes 130, 153Macrophages 5–6, 59iron-storage 57, 58, 136role in erythropoiesis 30, 32Malaria 19, 158–161

196IndexMalignant lymphogranulomatosis 70Malignant transformations 8Mastocytosis 125, 126May-Hegglin anomaly 41, 166, 168Mean cell hemoglobin content (MCH)10, 13, 128Mean cell volume (MCV) 10, 13Mean cellular hemoglobin concentration(MCHC) 10, 11, 13Measles 66, 166, 168Medulloblastoma 188, 189Megakaryocytes 50–51, 57diagnostic implications 50, 56in chronic myeloid leukemia 115,118in essential thrombocythemia170–171in hyperchromic anemia 154in iron deficiency 136in osteomyelosclerosis 122Megaloblasts 154Megalocytes 130, 152, 153Meningitis 188, 189Merozoites 158Metabolite deficiencies 7Metamyelocytes 4, 31, 35–37, 57, 153diagnostic implications 36Metastasesbone 150, 168lymph nodes 182, 183Microangiopathy 144thrombocytopenia in 166Microcytes 132, 133Micromegakaryocytes 118Micromyeloblasts 34Microspherocytosis 144, 145Monocytes 3, 41, 46–47acute monocytic leukemia 100diagnostic implications 46, 56functions 5–6normal ranges and mean values 12Monocytopoiesis 3in acute myelomonocytic leukemia98Monocytosis 46, 88–89causes 88Mononucleosis 66, 68–69, 168, 176,178Multiple myeloma (MM) 82, 83, 85Mumps 166Myeloblasts 4, 31, 34–35acute erythroleukemia 100acute myeloblastic leukemia 96–97diagnostic implications 34see also Blast crisisMyelocytes 4, 36–37, 57diagnostic implications 36in hyperchromic anemia 154Myelodysplasia (MDS) 40, 42,106–109, 131, 149bone marrow analysis 54, 108, 109classification 106, 108differential diagnosis 154–155Myelofibrosis (MF) 26Myelomamultiple (MM) 82, 83, 85plasma cell 71, 82, 84Myeloproliferative diseases 32, 44, 50,56, 149basophilia and 124–126chronic myeloproliferative disorders(CMPD) 114–115differential diagnosis 115see also specific diseasesNNatural killer (NK) cells 48Neutropenia 86autoimmune 86classification 86congenital/familial 86secondary, in bone marrow disease86Neutrophilia 110causes 111, 112reactive left shift 112–113without left shift 110Neutrophils 12, 31, 38–41, 45cerebrospinal fluid 188functions 5, 6segmented 38–41, 43diagnostic implications 38normal ranges and mean values12see also Neutropenia; NeutrophiliaNon-Hodgkin lymphoma (NHL) 26,70–73blastic 70cell surface markers 72–73lymph node biopsy 182, 183Normal values 15–17Normoblasts 32, 138, 150

Index197in hemolytic anemia 140orthochromatic 140Nuclear appendages 42, 43OOsteoblasts 58, 59Osteoclasts 58, 59Osteomyelosclerosis (OMS) 26, 111,114, 122–123characteristics 122Oversegmentation 38PPancytopenia 80Panmyelopathy 56, 148–149causes 149Panmyelophthisis 8, 148–150, 168Parasite infections 44, 124, 188eosinophilia and 124, 188Pel–Ebstein fever 40Pelger anomaly 40Perls' Prussion blue reaction 56Pertussis 66Pfeiffer cells 68, 69Philadelphia chromosome 114, 115,116Plasma cells 48–49, 57, 77, 82–85counts 56diagnostic implications 48, 82Plasmablasts 176Plasmacytoma 56, 70, 71, 82–85, 149morphological variability 84staging 84Plasmodia 19, 158–161Pleural effusions 186–187Poikilocytosis 130, 134, 135, 137–139,152Polychromasia 135Polychromophilia 134, 137Polycythemia 8, 53, 56, 111, 131erythremic 162–163Polycythemia vera (PV) 114, 122,162–163diagnosis 162rubra 26Proerythroblasts 30–31, 54, 85Promyelocytes 4, 31, 34–35, 44, 57acute promyelocyte leukemia 98, 99diagnostic implications 34, 83Pseudo Gaucher cells 118Pseudo-Pelger formation 40, 41, 107diagnostic implications 42, 118Pseudopolycythemia 53Pseudothrombocytopenia 15, 51, 164,166–167Q5q-syndrome 108RRabbit fever 179Reactive lymph node hyperplasia 176,177Reactive lymphocytosis 66–67Red cell distribution width (RDW) 11Reed–Sternberg giant cells 176, 177Reference ranges 15–17Refractive anemia with excess blasts(RAEB) 54Renal insufficiency 146Reticular cells, fibroblastic 58, 59Reticulocytes 32counts 11, 13, 32, 128in hemolytic anemia 140–141normal ranges and mean values 13Reticuloendothelial system (RES), ironpull 134, 136Richter syndrome 74, 77Rubella 66, 166, 168, 178Russell bodies 84, 85SSarcoidosis 178, 180–181Scarlet fever 40Schistocytosis 144Schizocytes 142Schizonts 158Schüffner's dots 158Scrofuloderma 180Sepsis 40, 41, 113, 167Sézary syndrome 74, 77Sickle cells 142, 144, 145Sideroachresia 56, 137Sideroblasts 56, 137

198IndexSideroblasts, ring 56, 106, 109, 137Siderophages 188Spherocytosis 142Splenic lymphoma with villouslymphocytes (SLVL) 80, 81Splenomegaly 80, 112, 114–116, 142in chronic myeloid leukemia114–115, 116in osteomyelosclerosis 123Staff cells 4Staining 18, 19iron staining of erythropoietic cells56, 57, 109Stem cells 2–3Still disease 179Stomatocytes 144, 145Stomatocytosis 144Strongyloides stercoralis 124Subarachnoid hemorrhage 188, 189Substantia granulofilamentosa 156TT-lymphocytes 48function 6see also LymphocytesTarget cells 130, 138, 139, 142Thalassemia 131, 138–139, 142major 138, 139minor 138, 139Thrombocytes 7, 33, 50–51, 165–169counts 15, 25diagnostic implications 50function 7normal ranges and mean values 13Thrombocythemiaessential (ET) 8, 26, 56, 114,170–171diagnosis 170idiopathic 122Thrombocytopenia 8, 56, 80, 113,164–169due to increased demand 164–167due to reduced cell production168–169in chronic myeloid leukemia 121in hypersplenism 166in microangiopathy 166in myelodysplasia 107Thrombocytopenic purpura (TTP) 131,144, 166post-transfusion 166Thrombocytosis 170–171reactive 170Thrombopoiesis 3Tissue mast cells 125, 126Toxic effects 8anemia 134Toxic granulation 40–41, 113diagnostic implications 42Toxoplasmosis 176, 178, 180lymphomatous 66Tricytopenia 102, 106, 149Trophozoites 158, 161Tuberculosis 178, 180–181Tularemia 179Tumorsanemia and 134biopsy 23see also specific tumorsUUrticaria pigmentosa 126VVacuoles 40, 41Virocytes 66, 68, 69Vitamin B12 deficiency 7, 38, 54, 149in hyperchromic anemia 152–154thrombocytopenia and 168WWaldenström syndrome 78Werlhof syndrome 56Wheel-spoke nuclei 84Whooping cough 66Wiscott-Aldrich syndrome 168XX-chromosome 42, 43ZZieve syndrome 142